Process of preparing guanylate cyclase c agonisys

ABSTRACT

The invention provides processes of preparing a peptide including a GCC agonist sequence selected from the group consisting of SEQ ID NOs: 1-249 described herein.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalApplication No. 61/447,891 filed on Mar. 1, 2011, the content of whichis incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to processes of preparing guanylatecyclase C peptide agonists useful for the treatment and prevention ofvarious diseases and disorders.

BACKGROUND OF THE INVENTION

Guanylate cyclase C is a transmembrane form of guanylate cyclase that isexpressed on various cells, including gastrointestinal epithelial cells(reviewed in Vaandrager 2002 Mol. Cell. Biochem. 230:73-83). It wasoriginally discovered as the intestinal receptor for the heat-stabletoxin (ST) peptides secreted by enteric bacteria and which causediarrhea. The ST peptides share a similar primary amino acid structurewith two peptides isolated from intestinal mucosa and urine, guanylinand uroguanylin (Currie, et al., Proc. Nat'l Acad. Sci. USA 59:947-951(1992); Hamra, et al., Proc. Nat l Acad. Sci. USA 90: 10464-10468(1993); Forte, L., Reg. Pept. 57:25-39 (1999); Schulz, et al., Cell55:941 -948 (1990); Guba, et al., Gastroenterology 777: 1558-1568(1996); Joo, et al., Am. J. Physiol. 27:G633-G644 (1998)).

In the intestines, guanylin and uroguanylin act as regulators of fluidand electrolyte balance. In response to high oral salt intake, thesepeptides are released into the intestinal lumen where they bind toguanylate cyclase C localized on the luminal membrane of enterocytes(simple columnar epithelial cells of the small intestines and colon).The binding of the guanylin peptides to guanylate cyclase C induceselectrolyte and water excretion into the intestinal lumen via a complexintracellular signaling cascade that is initiated by an increase incyclic guanosine monophosphate (cGMP).

The cGMP-mediated signaling that is initiated by the guanylin peptidesis critical for the normal functioning of the gut. Any abnormality inthis process could lead to gastrointestinal disorders such as irritablebowel syndrome (IBS) and inflammatory bowel diseases. Inflammatory boweldisease is a general name given to a group of disorders that cause theintestines to become inflamed, characterized by red and swollen tissue.Examples include ulcerative colitis and Crohn's disease. Crohn's diseaseis a serious inflammatory disease that predominantly affects the ileumand colon, but can also occur in other sections of the gastrointestinaltract. Ulcerative colitis is exclusively an inflammatory disease of thecolon, the large intestine. Unlike Crohn's disease, in which all layersof the intestine are involved, and in which there can be normal healthybowel in between patches of diseased bowel, ulcerative colitis affectsonly the innermost lining (mucosa) of the colon in a continuous manner.Depending on which portion of the gastrointestinal tract is involved,Crohn's disease may be referred to as ileitis, regional enteritis,colitis, etc. Crohn's disease and ulcerative colitis differ from spasticcolon or irritable bowel syndrome, which are motility disorders of thegastrointestinal tract. Gastrointestinal inflammation can be a chroniccondition. It is estimated that as many as 1,000,000 Americans areafflicted with inflammatory bowel disease, with male and female patientsappearing to be equally affected. Most cases are diagnosed before age30, but the disease can occur in the sixth, seventh, and later decadesof life.

IBS and chronic idiopathic constipation are pathological conditions thatcan cause a great deal of intestinal discomfort and distress but unlikethe inflammatory bowel diseases, IBS does not cause the seriousinflammation or changes in bowel tissue and it is not thought toincrease the risk of colorectal cancer. In the past, inflammatory boweldisease, celiac disease, and IBS were regarded as completely separatedisorders. Now, with the description of inflammation, albeit low-grade,in IBS, and of symptom overlap between IBS and celiac disease, thiscontention has come under question. Acute bacterial gastroenteritis isthe strongest risk factor identified to date for the subsequentdevelopment of postinfective irritable bowel syndrome. Clinical riskfactors include prolonged acute illness and the absence of vomiting. Agenetically determined susceptibility to inflammatory stimuli may alsobe a risk factor for irritable bowel syndrome. The underlyingpathophysiology indicates increased intestinal permeability andlow-grade inflammation, as well as altered motility and visceralsensitivity. Serotonin (5-hydroxytryptamine [5-HT]) is a key modulatorof gut function and is known to play a major role in pathophysiology ofIBS. The activity of 5-HT is regulated by cGMP.

While the precise causes of IBS and inflammatory bowel diseases (IBD)are not known, a disruption in the process of continual renewal of thegastrointestinal mucosa may contribute to disease pathology in IBD andaggravate IBS. The renewal process of the gastrointestinal lining is anefficient and dynamic process involving the continual proliferation andreplenishment of unwanted damaged cells. Proliferation rates of cellslining the gastrointestinal mucosa are very high, second only to thehematopoietic system. Gastrointestinal homeostasis depends on both theproliferation and programmed cellular death (apoptosis) of epithelialcells lining the gut mucosa. Cells are continually lost from thevillU.S. Pat. No. into the lumen of the gut and are replenished at asubstantially equal rate by the proliferation of cells in the crypts,followed by their upward movement to the villU.S. Pat. No. . The ratesof cell proliferation and apoptosis in the gut epithelium can beincreased or decreased in a variety of circumstances, e.g., in responseto physiological stimuli such as aging, inflammatory signals, hormones,peptides, growth factors, chemicals and dietary habits. In addition, anenhanced proliferation rate is frequently associated with a reduction inturnover time and an expansion of the proliferative zone. Theproliferation index is much higher in pathological states such asulcerative colitis and other gastrointestinal disorders. Intestinalhyperplasia is a major promoter of gastrointestinal inflammation.Apoptosis and cell proliferation together regulate cell number anddetermine the proliferation index. Reduced rates of apoptosis are oftenassociated with abnormal growth, inflammation, and neoplastictransformation. Thus, both increased proliferation and/or reduced celldeath may increase the proliferation index of intestinal tissue, whichmay in turn lead to gastrointestinal inflammatory diseases.

In addition to a role for uroguanylin and guanylin as modulators ofintestinal fluid and ion secretion, these peptides may also be involvedin the continual renewal of gastrointestinal mucosa by maintaining thebalance between proliferation and apoptosis. For example, uroguanylinand guanylin peptides appear to promote apoptosis by controllingcellular ion flux. Given the prevalence of inflammatory conditions inWestern societies a need exists to improve the treatment options forinflammatory conditions, particularly of the gastrointestinal tract.

Peptide agonists of guanylate cyclase C agonists (“GCC agonists”) aredescribed in U.S. Pat. Nos. 7,041 ,786, 7,799,897, and U.S. PatentApplication Publication Nos. U.S. PAT. NO. 2009/0048175, U.S. PAT. NO.2010/0069306, U.S. PAT. NO. 2010/0120694, U.S. PAT. NO. 2010/0093635,and U.S. PAT. NO. 2010/0221329. However, the solid phase synthesis ofpeptides for pharmaceutical application presents a number of specialproblems such as an overall low yield (e.g., less than 10%).

SUMMARY OF THE INVENTION

The present invention provides a process of preparing a peptide,particularly a peptide comprising the sequence of a peptide agonist ofguanylate cyclase C (“GCC”). The GCC agonist sequence is selected fromthe group consisting of SEQ ID NOs: 1-249. The GCC agonist sequence is namino acid units in length, with the N-terminal unit at position 1 andthe C-terminal unit at position n.

The process of the invention includes solid phase and/or solution phasesyntheses of suitable peptide fragments, subsequent fragmentcondensation in a solution to form a linear crude peptide, and optionaloxidative cyclization of cysteine amino acid residues of the linearcrude peptide to form the cyclized final product. Particularly, theprocess includes the following steps:

-   -   providing a first fragment having a first sequence of amino acid        units from position j through position k of the GCC agonist        sequence, wherein y is an integer between 1 and n−1, k is an        integer between 2 and n and is greater than j, and the first        fragment is protected except for an amino group of the amino        acid unit at position j, or alternatively, a carboxyl group of        the amino acid unit at position k,    -   providing a second fragment having a second sequence of amino        acid units from position h through position j−1 of the GCC        agonist sequence or having a third sequence of amino acid units        from position k+1 through position m of the GCC agonist        sequence, wherein h is an integer between 1 and n−2 and is        smaller than j, m is an integer between k+2 and n, and the        second fragment is protected except for a carboxyl group of the        amino acid unit at position j−1 or an amino group of the amino        acid unit at position k+1, and    -   coupling the first and the second fragments via a solution-phase        synthesis to yield a protected peptide having a sequence of        amino acid units from position h through position k of the GCC        agonist sequence or a protected peptide having a sequence of        amino acid units from position j through position m of the GCC        agonist sequence.

The process of the invention may include one or more of the featuresdescribed in the following embodiments.

In one embodiment, the GCC agonist sequence is selected from the groupconsisting of SEQ ID NOs: 1, 8, 9, 55, 56, 58, and 59.

In one embodiment, the GCC agonist sequence is SEQ ID NO: 1 or 9. In oneembodiment, h is 1, j is 7, and k is 16. More specifically, the firstfragment has a sequence of amino acid units from position 7 throughposition 16 of SEQ ID NO: 1 or 9 and the second fragment has a sequenceof amino acid units from position 1 through position 6 of SEQ ID NO: 1or 9. In another embodiment, h is 7, j is 15, and k is 16. Morespecifically, the first fragment has a sequence of amino acid units fromposition 15 through position 16 of SEQ ID NO: 1 or 9, the secondfragment has a sequence of amino acid units from position 7 throughposition 14 of SEQ ID NO: 1 or 9, and the protected peptide produced viacoupling the first and second fragments has a sequence of amino acidunits from position 7 through position 16 of SEQ ID NO: 1 or 9. In oneembodiment, the process of the invention further includes deprotectingan amino group of the amino acid unit at position 7 of the protectedpeptide having the sequence of amino acid units from position 7 throughposition 16 of SEQ ID NO: 1 or 9 to yield a position-7 reactive peptide.In one embodiment, the process of the invention further includesproviding a third fragment having a sequence of amino acid units fromposition 1 through position 6 of SEQ ID NO: 1 or 9, wherein the thirdfragment is protected except for a carboxyl group of the amino acid atposition 6. In one embodiment, the process further includes coupling thethird fragment and the position-7 reactive peptide via a solution-phasesynthesis to yield a protected linear peptide having a sequence of aminoacid units from position 1 through position 16 of SEQ ID NO: 1 or 9. Inone embodiment, the process further includes deprotecting the protectedlinear peptide to yield a deprotected linear peptide. In one embodiment,the process further comprises oxidizing the deprotected linear peptideto yield the peptide having the GCC agonist sequence of SEQ ID NOs: 1 or9.

In one embodiment, each of the first and second fragments is not morethan 10 amino acid units in length (e.g., 2-10 amino acid units inlength, 3-9 amino acid units in length, or 4-8 amino acid units inlength).

In one embodiment, the second fragment has the second sequence of aminoacid units from position h through position j−1 of the GCC agonistsequence and either one or both of the amino acid unit of the secondfragment at position j−1 and the amino acid unit of the first fragmentat position k is selected from the group consisting of glycine, proline,leucine, alanine, and arginine. In one embodiment, at least one of theamino acid units at positions j−1 and k is either glycine or proline.

In one embodiment, the second fragment has the third sequence of aminoacid units from position k+1 through position m of the GCC agonistsequence and either one or both of the amino acid unit of the thirdfragment at position m and the amino acid unit of the first fragment atposition k is selected from the group consisting of glycine, proline,leucine, alanine, and arginine. In one embodiment, at least one of theamino acid units at positions m and k is either glycine or proline.

In one embodiment, k is n. In one embodiment, the second fragment hasthe second sequence of amino acid units from position h through positionj−1 of the GCC agonist sequence, and the amino acid unit at positionsj−1 is selected from the group consisting of glycine, proline, leucine,alanine, and arginine. In one embodiment, the amino acid unit atposition j−1 is either glycine or proline. In one embodiment, theprocess of the invention further comprises deprotecting an amino groupof the amino acid unit at position h of the protected peptide having asequence of amino acid units from position h through position k of theGCC agonist sequence to yield a position-h reactive peptide. In oneembodiment, the process further comprises providing a fourth fragmenthaving a sequence of amino acid units from position 1 through positionh−1 of the GCC agonist sequence, wherein the fourth fragment isprotected except for a carboxyl group of the amino acid unit at positionh−1. In one embodiment, the fourth fragment is not more than 10 aminoacid units in length (e.g., 2-10 amino acid units in length, 3-9 aminoacid units in length, or 4-8 amino acid units in length). In oneembodiment, the process further includes coupling the fourth fragmentand the position-h reactive peptide via a solution-phase synthesis toyield a protected linear peptide having a sequence of amino acid unitsfrom position 1 through position n of the GCC agonist sequence. In oneembodiment, the process further comprises deprotecting the protectedlinear peptide to yield a deprotected linear peptide having a sequenceof amino acid units from position 1 through position n of the GCCagonist sequence. In one embodiment, the process of the inventionfurther comprises oxidizing the deprotected linear peptide to yield thepeptide comprising the GCC agonist sequence selected from the groupconsisting of SEQ ID NOs: 1-249.

In one embodiment, at least one of the fragments of the peptide ofinterest (e.g., the first, second, third, and/or fourth fragments) isprovided via a solid-phase peptide synthesis. In one embodiment, thesolid-phase peptide synthesis is a Fmoc solid-phase synthesis. In oneembodiment, the Fmoc solid-phase synthesis is performed on2-chlorotrityl resin, such as those having 1% DVB and a substitutionrate ranging from 0.3 mmol/g to 1.2 mmol/g (e.g., 0.9-1.1 mmol/g).

The methods of this invention unexpectedly produce GCC agonist peptidesof high purities (e.g., >96%) at high yields (e.g., >14%) compared toconventional step-by-step solid-phase peptide synthesis (SPPS), wherethe overall yield of the peptides having a 96% purity level isapproximately 5%. The fragment condensation process (i.e., hybridsolution- and solid-phase process) of this invention also requires muchless time for synthesizing GCC peptides than conventional SPPS. Inaddition to the cost effectiveness afforded by the increased yields andreduced time, the process of this invention is also readily scalable forcommercial production.

In another aspect, the invention features the peptides, e.g., GCCagonist peptides, prepared by the methods described herein.

In still another aspect, the invention also provides a process ofpurifying the peptide comprising the GCC agonist sequence selected fromthe group consisting of SEQ ID NOs: 1-249. The process comprisesadsorbing the peptide onto a polymeric adsorbent column, optionallyrinsing the peptide with deionized water, eluting the optionally rinsedpeptide off the polymeric adsorbent column with a solvent mixture (e.g.,an alcohol aqueous solution) to form a peptide solution, removing partor all of the solvent mixture (e.g., water and the alcohol) from thepeptide solution to precipitate the peptide, and optionally adding anether to the dewatered peptide to facilitate precipitation of thepeptide.

In one embodiment, the peptide is precipitated by concentrating thepeptide solution by removing water and the alcohol under vacuum withoutthe need of adding an ether. In another embodiment, an ether is added tofacilitate the precipitation, e.g., by hastening the precipitationprocess.

In one embodiment, rinsing the peptide after adsorption onto a polymericadsorbent column is not needed when, e.g., the peptide adsorbed issubstantially free of water-soluble salts (e.g., phosphates oracetates). In this context “substantially” free of water-soluble saltsmeans that the salt content of the peptide is preferably less than 5%,less than 4.5%, less than 4.25%, less than 4%, less than 3.5%, less than3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, lessthan 0.5%, less than 0.25%, or less than 0.1 %, of the total weight ofthe peptide.

In one embodiment, the polymeric adsorbent column comprises apolystyrene resin. In particular, the resin is selected so that thepurified peptide eluted or desorbed is not less than 80% of the peptideamount adsorbed on the resin, e.g., not less than 85%, not less than90%, or not less than 95%. In one embodiment, the resin is formed ofcrosslinked polystyrene with an average pore diameter greater than 5 nm,e.g., about 6-8 nm, 10-15 nm, 15-20 nm, or 25-30 nm.

In one embodiment, the solvent mixture used for eluting the peptidecomprises water, e.g., a mixture of water and one or more secondsolvents which can form azeotrope with water such as ethanol,isopropanol, tert-butanol, 2-butanol, 1-chloro-2-propanol,1-methoxy-2-propanol, 2-methoxy-ethanol, 2-methyl-2-propanol, aceticacid, methyl acetate, and propyl acetate. The one or more secondsolvents can be Class 3 solvents (or low-toxicity solvents) as definedin the ICH guideline. In another embodiment, the solvent mixture usedfor eluting comprises ether, such as a mixture of ethanol/ether orisopropanol/ether

In one embodiment, the alcohol aqueous comprises isopropanol, propanol,tert-butanol, 2-butanol, or ethanol.

In one embodiment, the ether comprises diethyl ether or MTBE.

In one embodiment, the process further includes salt exchanging thepeptide by washing the peptide with an aqueous solution comprising anammonium salt (e.g., ammonium acetate), acetic acid, and/or an acetatesalt (e.g., sodium acetate) before the purification. In one embodiment,if the process includes the salt exchanging step, the process alsoincludes rinsing the peptide with deionized water after adsorption onthe polymeric resin column to desalt the peptide.

In one embodiment, the process further includes lyophilizing the peptideafter the salt exchanging step before the purification.

In one embodiment, the process further includes drying the precipitatedpeptide after adding the ether, e.g., under reduced pressure.

In one embodiment, the purified peptide is selected from the groupconsisting of SEQ ID NOs: 1, 8, 9, and 56. Preferably, the purifiedpeptide is SEQ ID NO: 1 or 9.

The purification methods of this invention unexpectedly produce GCCagonist peptides of high purities (e.g., >96%) and high bulk and/or tapdensities (e.g., >0.3 g/mL) compared to conventional purificationmethods such as lyophilization, where the bulk and/or tap densities ofthe purified are at approximately 10 times lower than those of thepeptides purified by the methods of this invention. The purificationprocess of this invention also requires much less time than conventionallyophilization, which can potentially lead to reductions in topoisomercontent, deamidation degradation products, and other impurities. Thepurification process of this invention is also readily scalable forcommercial production compared to conventional lyophilization.

In yet another aspect, the invention features the peptides, e.g., GCCagonist peptides, purified by the process described herein. The purifiedpeptides may have one or more of the following features.

In one embodiment, the purified peptide has a bulk density of no lessthan 0.05 g/mL, no less than 0.1 g/mL, no less than 0.2 g/mL, no lessthan 0.3 g/mL, no less than 0.4 g/mL, or no less than 0.5 g/mL. Forexample, the purified peptide has a bulk density ranging between 0.05g/mL and 2 g/mL.

In one embodiment, the purified peptide has a tap density of no lessthan 0.08 g/mL, no less than 0.1 g/mL, no less than 0. 15 g/mL, no lessthan 0.2 g/mL, no less than 0.3 g/mL, no less than 0.4 g/mL, no lessthan 0.5 g/mL, or no less than 0.6 g/mL. For example, the purifiedpeptide has a tap density ranging between 0.08 g/mL and 2 g/mL.

In one embodiment, the purified peptide has a chromatographic purity ofno less than 96%, no less than 97%, or no less than 98%. For example,the GCC agonist peptide has chromatographic impurity content of nogreater than 4%, no greater than 3.5%, no greater than 3%, no greaterthan 2.5%, no greater than 2%, no greater than 1.5%, or no greater than1%. The chromatographic impurity content is determined as total areapercentages of impurities by HPLC. The chromatographic impurity contentincludes topoisomer content. The impurities do not include anypharmaceutically acceptable excipient used for drug formulation.

In one embodiment, the purified peptide is substantially free ofcontaminants resulted from the peptide preparation process such asorganic solvents used in the process, e.g., ammonium, acetonitrile,acetamide, alcohol (e.g., methanol, ethanol, or isopropanol), TFA, etheror other contaminants. In this context “substantially” free ofcontaminants means that the contaminant content of the peptide at theend of the purification process is preferably less than 0.5%, less than0.3%, less than 0.25%, less than 0.1 %, less than 0.05%, less than0.04%, less than 0.03%, less than 0.02%, less than 0.01 %, less than0.005%, less than 0.003%, or less than 0.001 % of the total weight ofthe peptide. For example, the purified peptide contains <0.01%acetamide, <0.3% ammonium ion, <0.01 % acetonitrile, and/or <0.1 % TFA.The content of contaminants can be determined by conventional methodssuch as gas chromatography. Preferably, the residual solvents in thepurified peptide of the invention are less than the limits set in theICH guidelines, e.g., IMPURITIES: GUIDELINE FOR RESIDUAL SOLVENTSQ3C(R5) (available at

http://www.ich.Org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3C/Step4/Q3C_R5_Step4.pdf). For example, the purified peptide contains <410 ppmacetonitrile (e.g., ≦40 ppm or ≦20 ppm), <5000 ppm ethanol (e.g., ≦140ppm), <5000 ppm isopropanol, <5000 ppm ethyl acetate (e.g., ≦20 ppm),<3000 ppm methanol (e.g., ≦250 ppm), <5000 ppm MTBE (e.g., ≦20 ppm),<290 ppm hexane, and/or <5000 ppm heptane or pentane.

In one embodiment, the purified peptide is substantially free oftopoisomers. In this context “substantially” free of topoisomers meansthat the topoisomer content of the peptide at the end of thepurification process is preferably less than 2%, less than 1.5%, lessthan 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than0.7%, less than 0.6%, less than 0.5%, less than 0.4%), less than 0.3%,less than 0.2%, or less than 0.1%, of the total weight of the peptide.

In one embodiment, the purified peptide is substantially free of water.In this context “substantially” free of water means that the watercontent of the peptide at the end of the purification process ispreferably less than 7%, less than 6%, less than 5%, less than 4.5%,less than 4.25%, less than 4%, less than 3.5%, less than 3%, less than2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, lessthan 0.25%, or less than 0.1%, of the total weight of the peptide.

In one embodiment, the purified peptide includes the GCC agonistsequence of SEQ ID NO: 1.

In one embodiment, the purified peptide includes the GCC agonistsequence of SEQ ID NO: 9.

The invention also relates to a formulation (e.g., an oral formulation)containing the peptides prepared and/or purified by the methodsdescribed herein and in particular, a low dose formulation containing0.05-10 mg (e.g., 0.1 mg, 0.3 mg or 0.5 mg) of the purified peptides.The low-dose formulation can further have one or more additionalfeatures as described in PCT/U.S. PAT. NO. 2011 /051805 and can beprepared by the methods disclosed therein, such as dry blending.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing particle size distribution by sieving analysisfor lyophilized plecanatide and precipitated plecanatide.

FIG. 2 is an optical microscopic image of lyophilized plecanatide.

FIG. 3 is an optical microscopic image of precipitated plecanatide.

DETAILED DESCRIPTION

The invention provides processes of preparing peptides, e.g., peptideGCC agonists, in particular a hybrid solution- and solid-phase process.The process of the invention includes providing two or more fragments ofa peptide of interest via solid-phase and/or solution-phase synthesesand coupling them via a solution-phase synthesis to obtain the targetpeptide. The process may further include, if needed, oxidativecyclization of cysteine amino acid residues of a linear peptide formedby the fragment coupling to produce a cyclized peptide.

The fragments described above can be prepared by standard solution phasepeptide synthesis or solid phase peptide synthesis techniques in which apeptide linkage occurs through the direct condensation of the aminogroup (i.e., NH₂) of a first amino acid with the carboxy group (i.e.,COOH) of a second amino acid with the elimination of a water molecule.In one embodiment, at least one of the fragments is prepared by solidphase peptide synthesis.

Peptide bond synthesis by direct condensation, as formulated above,requires suppression of the reactive character of the amino group of thefirst and of the carboxyl group of the second amino acid. The maskingsubstituents (i.e., protecting groups) must permit their ready removal,without inducing breakdown of the labile peptide molecule. The term“protected peptide” or “protected peptide fragment” refers to a peptideor peptide fragment, in which all reactive groups on its constitutingamino acids, are masked by protecting groups, unless otherwisespecified. The term “deprotected peptide” or “deprotected peptidefragment” refers to a peptide or peptide fragment, in which all reactivegroups on its constituting amino acids, are free from being masked byprotecting groups, unless otherwise specified. The term “reactivegroups” refers to the groups forming the peptide bond and thoseinterfering with the peptide bond formation, such as amino, carboxyl,hydroxyl, and thiol (as in cysteine) groups. Examples of protectinggroups for amino include and are not limited to9-fluorenylmethyloxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), benzoyl(Bz), acetyl (Ac), and benzyl (Bn). Examples of protecting groups forcarboxyl include trityl (triphenylmethyl, Trt) and O-tert-butyl (OtBu).Examples of protecting groups for thiol include acetamidomethyl (Acm),tert-butyl (tBu), 3-nitro-2-pyridine sulfenyl (NPYS),2-pyridine-sulfenyl (Pyr), and trityl (Trt). Additional examples ofprotecting groups are described in Greene, T. W., Wuts, P. G. M.,Protective Groups in Organic Synthesis, 3^(rd) edition, John Wiley &Sons: New York, 1999, whose context is incorporated by reference herein.

In a preferred embodiment, the methods of the invention are used forpreparing SP-304 (plecanatide). In particular, three peptide fragments,A, B and C are prepared and then a linear peptide sequence is assembledby the condensation of fragment A, B and C as follows: preparingfragment A, Boc-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-Leu-OH,by solid phase from 2-chloro-trityl chloride resin; preparing fragmentB, Fmoc-Cys(Acm)-Val-Asn-Val-Ala-Cys(Trt)-Thr(tBu)-Gly-OH, by solidphase from 2-chlorotrityl chloride resin; preparing fragment C,Cys(Acm)-Leu-OtBu, by solution phase synthesis, coupling fragments B andC in solution phase to yield fragment B-C, and coupling fragments A andB-C to yield linear peptide A-B-C.

The side-chain-protected Fragments A (BocAA 1 -60H) and B (FmocAA7-140H)can be prepared by Fmoc SPPS using the super acid-sensitive2-chlorotrityl chloride (2-ClTrt) resin and Fmoc-protected amino acidderivatives, as shown in Scheme 1 below.

Fragment C (HAA15-16OtBu) can be prepared by the solution phasesynthesis and then be coupled to Fragment B (FmocAA7-14OH) in solutionphase to give Fragment B-C (FmocAA7-16OtBu). The Fmoc protecting groupcan then be removed from Fragment B-C (FmocAA7-16OtBu) to giveHAA7-16OtBu, which is then coupled to Fragment A (BocAA1-6OH) to yieldside-chain-protected linear SP-304 (BocAA1-16OtBu), as shown in Scheme 2below.

The side-chain-protected linear SP-304 (BocAA1-16OtBu) can be treatedwith trifluoroacetic acid/triisopropylsilane/ethanedithiol (TFA/TIS/EDT)to give the partially protected SP-304 (HAA1-16OH) in which the 2 S-Acmgroups (as shown in Scheme 2 above) are intact. The partially protectedlinear SP-304 (HAA1-16OH) can be oxidized by H₂O₂, followed bysimultaneous removal of the S-Acm groups and disulfide formation withiodine to give crude dicyclic SP-304, as shown in Schemes 3 and 4 below.

The solution of crude dicyclic SP-304 can then be purified andconcentrated, as shown in Scheme 3 above, by loading the solution to apolystyrenic adsorbent resin (e.g., D101 (Anhui Sanxing (China);crosslinked polystyrene; surface area 500-550 m²/g; average porediameter: 9-10 nm; pore volume: 1.18-1.24 ml/g; bulk density: 0.65-0.70g/ml; specific density: 1.03-1.07 g/ml; moisture: 67-75%; particle size:0.315-1.25 mm≧95%; effective diameter: 0.4-0.7 mm; uniformitycoefficient: ≦1.6%), DA201C, DA201H, ADS-8, and ADS-5) column, elutingthe dicyclic SP-304 from the column with an eluent (e.g., a 90% ethanolaqueous solution), concentrating the collected SP-304 solution underreduced pressure, and precipitating SP-304 with methyl t-butyl ether(MTBE). The precipitate can then be collected by filtration orcentrifugation, dried under high vacuum to give SP-304 in solid form.

As illustrated in Scheme 4 above, the solution of crude dicyclic SP-304can also be purified directly on preparative HPLC C18 column withacetonitrile (ACN), methanol, and/or water in various buffer system. Thecrude dicyclic SP-304 can also be purified via other methods known to askilled person in the art.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection, and coupling reactions must go to completionand the side-chain blocking groups must be stable throughout thesynthesis.

Acetylation of the N-terminal can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (See, Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful fragment selection is necessary to minimize racemization duringfragment condensation. For example, racemization is minimized whenfragments contain C-terminal Gly or Pro. Solubility considerations arealso a factor. Solid phase peptide synthesis uses an insoluble polymerfor support during organic synthesis. The polymer-supported peptidechain permits the use of simple washing and filtration steps instead oflaborious purifications at intermediate steps. Solid-phase peptidesynthesis may generally be performed according to the method ofMerrifield et al., J. Am. Chem. Soc, 1 963, 85:2149, which involvesassembling a linear peptide chain on a resin support using protectedamino acids. Solid phase peptide synthesis typically utilizes either theBoc or Fmoc strategy, both of which are well known in the art.

The processes of the present invention can be used to make anypeptide-based GCC agonist known in the art, such as analogs ofuroguanylin, guanylin, lymphoguanylin, linaclotide, ST peptides, SP-304,and SP-333. Non-limiting examples of such analogs of uroguanylin,guanylin, lymphoguanylin, linaclotide, SP-304, SP-333, and bacterial STpeptides are described in Section 1.1 below. In certain embodiments, themethods are used to prepare a peptide consisting essentially of an aminoacid sequence selected from SEQ ID NOs: 1-249. In a preferredembodiment, the peptide thusly made consists essentially of an aminoacid sequence selected from SEQ ID NOs: 1, 8, 9, 55, and 56. The term“consists essentially of” refers to a peptide that is identical to thereference peptide in its amino acid sequence or to a peptide that doesnot differ substantially in terms of either structure or function fromthe reference peptide. A peptide differs substantially from thereference peptide if its primary amino acid sequence varies by more thanthree amino acids from the reference peptide or if its activation ofcellular cGMP production is reduced by more than 50% compared to thereference peptide. Preferably, substantially similar peptides differ byno more than two amino acids and not by more than about 25% with respectto activating cGMP production. In preferred embodiments, the GCC agonistmade by the methods of the invention is a peptide comprising at least 12amino acid residues, and most preferably comprising between 12 and 26amino acids.

In another embodiment, the process of the invention is used forpreparing SP-353, which is a bacterial ST peptide analog. The generalstrategy for the hybrid synthesis of SP-353 includes solid phase andsolution phase syntheses to produce suitable peptide fragments (seeSchemes 5 and 6 below), subsequent segment condensation to form thelinear crude peptide (see Scheme 7 below), and natural oxidative foldingto form the cyclized final product (see Scheme 7 below). The samestrategy can also be used to produce other ST peptide analogs (such asSP-354, linaclotide, etc.) of similar amino acid sequences shown inTable II.

In yet another embodiment, the process of the invention is used forpreparing SP-333. The general strategy for the hybrid synthesis ofSP-333 includes solid phase and solution phase syntheses to producesuitable peptide fragments (see Schemes 8 and 9 below), subsequentsegment condensation to form the linear crude peptide (see Scheme 9below), oxidative folding to form the cyclized final product,purification, and lyophilization (see Scheme 10 below).

In another aspect, the invention also provides a process of purifyingpeptides, e.g., peptide GCC agonists. The general strategy for purifyingpeptides, including those synthesized by the hybrid methods disclosedherein, include, e.g., the steps illustrated in Scheme 11 below. It isunderstood that certain steps in Scheme 11 may be repeated (e.g.,rinsing column with deionized water) or absent (e.g., salt exchange oralcohol removal after dewatering) to optimize the purification process.

In yet another aspect, this invention provides a purified peptide, e.g.,peptide GCC agonists, purified by the precipitation process describedherein. Preferably, the purified peptide is SP-304 (SEQ ID NO:1) orSP-333 (SEQ ID NO:9). In one embodiment, the purified SP-304 or SP-333has a bulk density of no less than 0.05 g/mL, no less than 0.1 g/mL, noless than 0.2 g/mL, no less than 0.3 g/mL, no less than 0.4 g/mL, or noless than 0.5 g/mL. In a preferred embodiment, the purified SP-304 orSP-333 has a bulk density of about 0.05-2 g/mL, about 0.2-0.7 g/mL,about 0.3-0.6 g/mL, or about 0.4-0.5 g/mL. In one embodiment, thepurified SP-304 or SP-333 has a tap density of no less than 0.08 g/mL,no less than 0.1 g/mL, no less than 0.15 g/mL, no less than 0.2 g/mL, noless than 0.3 g/mL, no less than 0.4 g/mL, no less than 0.5 g/mL, or noless than 0.6 g/mL. For example, the purified SP-304 or SP-333 has a tapdensity of 0.08-2 g/mL, about 0.4-0.9 g/mL, about 0.5-0.8 g/mL, or about0.6-0.7 g/mL. In one embodiment, the purified peptide SP-304 or SP-333contains <0.01 % acetamide (e.g., <28 ppm), <0.3% ammonium ion (e.g.,<0.25%), <0.01 % acetonitrile (e.g., <20 ppm), and/or <0.1% TFA (e.g.,<0.09%). In one embodiment, the purified peptide SP-304 or SP-333 has abulk density of 0.4-0.5 g/mL, has a tap density of 0.6-0.7 g/mL, andcontains <0.01 % acetamide (e.g., <28 ppm), <0.3% ammonium ion (e.g.,<0.25%), <0.01 % acetonitrile (e.g., <20 ppm), and/or <0.1% TFA (e.g.,<0.09%).

1.1 GCC Agonists

The GCC agonists prepared by the processes of the invention can bind toguanylate cyclase C and stimulate intracellular production of cGMP.Optionally, the GCC agonists induce apoptosis and inhibit proliferationof epithelial cells. The term, “guanylate cyclase C” refers to atransmembrane form of guanylate cyclase that acts as the intestinalreceptor for the heat-stable toxin (ST) peptides secreted by entericbacteria. Guanylate cyclase C is also the receptor for the naturallyoccurring peptides guanylin and uroguanylin. The possibility that theremay be different receptors for each of these peptides has not beenexcluded. Hence, the term “guanylate cyclase C” may also encompass aclass of transmembrane guanylate cyclase receptors expressed onepithelial cells lining the gastrointestinal mucosa.

The term “GCC agonist” refers to both peptides and non-peptide compoundssuch as that bind to an intestinal guanylate cyclase C and stimulate theintracellular production of cGMP. Where the GCC agonist is a peptide,the term encompasses biologically active fragments of such peptides andpro-peptides that bind to guanylate cyclase C and stimulate theintracellular production of cGMP.

1.1.1 GCC Agonist Peptides

The GCC agonists prepared by the methods of the invention are preferablypeptides. In some embodiments, the GCC agonist peptide is less than 30amino acids in length. In particular embodiments, the GCC agonistpeptide is less than or equal to 30, 25, 20, 15, 14, 13, 12, 11, 10, or5 amino acids in length. Examples of GCC agonist peptides for U.S. Pat.No. e in the formulations and methods of the invention include thosedescribed in U.S. Pat. Nos. 7,879,802 and 8,034,782, and U.S.Publication Nos. 2010-0069306 and US2010-0120694, each of which isincorporated by reference herein in its entirety.

Specific examples of GCC agonist peptides that can be prepared by themethods of the invention include those described in Tables I-VII below.As used Tables I-VII, the terms “PEG3” or “3PEG” refer to a polyethyleneglycol such as aminoethyloxy-ethyloxy-acetic acid (AeeA), and polymersthereof. The term “X_(aa)” refers to any natural or unnatural amino acidor amino acid analogue. The term “M_(aa)” refers to a cysteine (Cys),penicillamine (Pen) homocysteine, or 3-mercaptoproline. The term“Xaa_(n1)” is meant to denote an amino acid sequence of any natural orunnatural amino acid or amino acid analogue that is one, two or threeresidues in length; Xaa_(n2) is meant to denote an amino acid sequencethat is zero or one residue in length; and Xaa_(n3) is meant to denotean amino acid sequence zero, one, two, three, four, five or six residuesin length. Additionally, any amino acid represented by Xaa, Xaa_(n1),Xaa_(n2), or Xaa_(n3) may be an L-amino acid, a D-amino acid, amethylated amino acid or any combination of thereof. Optionally, any GCCagonist peptide represented by Formulas I to XX in the tables maycontain one or more polyethylene glycol residues at the N-terminus,C-terminus or both.

In certain embodiments, a GCC agonist prepared by the methods of theinvention comprises a peptide selected from SEQ ID NOs: 1-249, thesequences of which are set forth below in Tables I to VII below. In oneembodiment, a GCC agonist prepared by the methods of the inventioncomprises the peptide designated by SEQ ID NOs: 1, 8, 9, 55, or 56.

In certain embodiments, a GCC agonist prepared by the methods of theinvention comprises a peptide that is substantially equivalent to apeptide selected from SEQ ID NOs: 1-249. The term “substantiallyequivalent” refers to a peptide that has an amino acid sequenceequivalent to that of the binding domain where certain residues may bedeleted or replaced with other amino acids without impairing thepeptide's ability to bind to an intestinal guanylate cyclase receptorand stimulate fluid and electrolyte transport.

In certain embodiments, the GCC agonist peptides are analogues ofuroguanylin or a bacterial ST peptide. Uroguanylin is a circulatingpeptide hormone with natriuretic activity. An ST peptide is a member ofa family of heat stable enterotoxins (ST peptides) secreted bypathogenic strains of E. coli and other enteric bacteria that activateguanylate cyclase receptor and cause secretory diarrhea. Unlikebacterial ST peptides, the binding of uroguanylin to guanylate cyclasereceptor is dependent on the physiological pH of the gut. Therefore,uroguanylin is expected to regulate fluid and electrolyte transport in apH dependent manner and without causing severe diarrhea.

The GCC agonist peptides prepared by the methods of the invention can bepolymers of L-amino acids, D-amino acids, or a combination of both. Forexample, in various embodiments, the peptides are D retro-inversopeptides. The term “retro-inverso isomer” refers to an isomer of alinear peptide in which the direction of the sequence is reversed andthe chirality of each amino acid residue is inverted. See, e.g., Jamesonet al., Nature, 368, 744-746 (1994); Brady et al., Nature, 368, 692-693(1994). The net result of combining D-enantiomers and reverse synthesisis that the positions of carbonyl and amino groups in each amide bondare exchanged, while the position of the side-chain groups at each alphacarbon is preserved. Unless specifically stated otherwise, it ispresumed that any given L-amino acid sequence of the invention may bemade into a D retro-inverso peptide by synthesizing a reverse of thesequence for the corresponding native L-amino acid sequence.

The GCC agonist peptides prepared by the methods of the invention areable to induce intracellular cGMP production in cells and tissuesexpressing guanylate cyclase C. In certain embodiments, the GCC agonistpeptide stimulates 5%, 10%, 20%, 30%, 40%, 50% , 75%, 90% or moreintracellular cGMP compared to naturally occurring GCC agonists such asuroguanylin, guanylin, or ST peptides. Optionally, the GCC agonistpeptide stimulates 5%, 10%, 20%, 30%, 40%, 50% , 75%, 90% or moreintracellular cGMP compared to SP-304 (SEQ ID NO: 1). In furtherembodiments, the GCC agonist peptide stimulates apoptosis, e.g.,programmed cell death, or activate the cystic fibrosis transmembraneconductance regulator (CFTR).

In some embodiments, the GCC agonist peptides prepared by the methods ofthe invention are more stable than naturally occurring GCC agonistsand/or SP-304 (SEQ ID NO: 1), SP-339 (linaclotide) (SEQ ID NO: 55) orSP-340 (SEQ ID NO: 56). For example, the GCC agonist peptide degrades2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50% , 75%, 90% or less compared tonaturally occurring GCC agonists and/or SP-304, SP-339 (linaclotide) orSP-340. In certain embodiments, the GCC agonist peptides for U.S. Pat.No. e in the formulations and methods of the invention are more stableto proteolytic digestion than naturally occurring GCC agonists and/orSP-304 (SEQ ID NO: 1), SP-339 (linaclotide) (SEQ ID NO: 55) or SP-340(SEQ ID NO: 56). In one embodiment, a GCC agonist peptide is pegylatedin order to render the peptides more resistant towards proteolysis byenzymes of the gastrointestinal tract. In a preferred embodiment, theGCC agonist peptide is pegylated with the aminoethyloxy-ethyloxy-aceticacid (Aeea) group at its C-terminal end, at its N-terminal end, or atboth termini.

Specific examples of GCC agonist peptides that can be prepared by themethods of the invention include a peptide selected from the groupdesignated by SEQ ID NOs: 1-249.

In one embodiment, the GCC agonist peptide is a peptide having the aminoacid sequence of any one of Formulas X-XVII (e.g. SEQ ID NOs: 87-98).

In some embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula I, wherein at least one amino acid ofFormula I is a D-amino acid or a methylated amino acid and/or the aminoacid at position 16 is a serine. Preferably, the amino acid at position16 of Formula I is a D-amino acid or a methylated amino acid. Forexample, the amino acid at position 16 of Formula I is a d-leucine or ad-serine. Optionally, one or more of the amino acids at positions 1-3 ofFormula I are D-amino acids or methylated amino acids or a combinationof D-amino acids or methylated amino acids. For example, Asn¹, Asp² orGlu^(J) (or a combination thereof) of Formula I is a D-amino acid or amethylated amino acid. Preferably, the amino acid at position Xa⁶ ofFormula I is a leucine, serine or tyrosine.

In alternative embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula II, wherein at least one amino acidof Formula II is a D-amino acid or a methylated amino acid. Preferably,the amino acid denoted by Xaa_(n2) of Formula II is a D-amino acid or amethylated amino acid. In some embodiments, the amino acid denoted byXaa_(n2) of Formula II is a leucine, a d-leucine, a serine, or ad-serine. Preferably, the one or more amino acids denoted by Xaa_(n1) ofFormula II is a D-amino acid or a methylated amino acid. Preferably, theamino acid at position Xaa⁶ of Formula II is a leucine, a serine, or atyrosine.

In some embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula III, wherein at least one amino acid ofFormula III is a D-amino acid or a methylated amino acid and/or aa isnot a cysteine. Preferably, the amino acid denoted by Xaa_(n2) ofFormula III is a D-amino acid or a methylated amino acid. In someembodiments the amino acid denoted by Xaa_(n2) of Formula III is aleucine, a d-leucine, a serine, or a d-serine. Preferably, the one ormore amino acids denoted by Xaa_(n1) of Formula III is a D-amino acid ora methylated amino acid. Preferably, the amino acid at position Xaa⁶ ofFormula III is a leucine, a serine, or a tyrosine.

In other embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula IV, wherein at least one amino acid ofFormula IV is a D-amino acid or a methylated amino acid, and/or Maa isnot a cysteine. Preferably, the Xaa_(n2) of Formula IV is a D-amino acidor a methylated amino acid. In some embodiments, the amino acid denotedby Xaa_(n2) of Formula IV is a leucine, a d-leucine, a serine, or ad-serine. Preferably, the one or more of the amino acids denoted byXaa_(n1) of Formula IV is a D-amino acid or a methylated amino acid.Preferably, the amino acid denoted Xaa⁶ of Formula IV is a leucine, aserine, or a tyrosine.

In further embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula V, wherein at least one amino acid ofFormula V is a D-amino acid or a methylated amino acid. Preferably, theamino acid at position 16 of Formula V is a D-amino acid or a methylatedamino acid. For example, the amino acid at position 16 (i.e., Xaa¹⁶) ofFormula V is a d-leucine or a d-serine. Optionally, one or more of theamino acids at position 1-3 of Formula V are D-amino acids or methylatedamino acids or a combination of D-amino acids or methylated amino acids.For example, Asn¹, Asp² or Glu³ (or a combination thereof) of Formula Vis a D-amino acids or a methylated amino acid. Preferably, the aminoacid denoted at Xaa⁶ of Formula V is a leucine, a serine, or a tyrosine.

In additional embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula VI, VII, VIII, or IX. Preferably, theamino acid at position 6 of Formula VI, VII, VIII, or IX is a leucine, aserine, or a tyrosine. In some aspects the amino acid at position 16 ofFormula VI, VII, VIII, or IX is a leucine or a serine. Preferably, theamino acid at position 16 of Formula V is a D-amino acid or a methylatedamino acid.

In additional embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula X, XI, XII, XIII, XIV, XV, XVI orXVII. Optionally, one or more amino acids of Formulas X, XI, XII, XIII,XIV, XV, XVI or XVII is a D-amino acid or a methylated amino acid.Preferably, the amino acid at the carboxy terminus of the peptidesaccording to Formulas X, XI, XII, XIII, XIV, XV, XVI or XVII is aD-amino acid or a methylated amino acid. For example the amino acid atthe carboxy terminus of the peptides according to Formulas X, XI, XII,XIII, XIV, XV, XVI or XVII is a D-tyrosine.

Preferably, the amino acid denoted by Xaa⁶ of Formula XIV is a tyrosine,phenylalanine or a serine. Most preferably the amino acid denoted byXaa⁶ of Formula XIV is a phenylalanine or a serine. Preferably, theamino acid denoted by Xaa⁴ of Formula XV, XVI or XVII is a tyrosine, aphenylalanine, or a serine. Most preferably, the amino acid positionXaa⁴ of Formula V, XVI or XVII is a phenylalanine or a serine.

In some embodiments, GCRA peptides include peptides containing the aminoacid sequence of Formula XVIII. Preferably, the amino acid at position 1of Formula XVIII is a glutamic acid, aspartic acid, glutamine or lysine.Preferably, the amino acid at position 2 and 3 of Formula XVIII is aglutamic acid, or an aspartic acid. Preferably, the amino acid atposition 5 a glutamic acid. Preferably, the amino acid at position 6 ofFormula XVIII is an isoleucine, valine, serine, threonine, or tyrosine.Preferably, the amino acid at position 8 of Formula XVIII is a valine orisoleucine. Preferably, the amino acid at position 9 of Formula XVIII isan asparagine. Preferably, the amino acid at position 10 of FormulaXVIII is a valine or a methionine. Preferably, the amino acid atposition 11 of Formula XVIII is an alanine. Preferably, the amino acidat position 13 of Formula XVII I is a threonine. Preferably, the aminoacid at position 14 of Formula XVIII is a glycine. Preferably, the aminoacid at position 16 of Formula XVIII is a leucine, serine or threonine

In alternative embodiments, GCRA peptides include peptides containingthe amino acid sequence of Formula XIX. Preferably, the amino acid atposition 1 of Formula XIX is a serine or asparagine. Preferably, theamino acid at position 2 of Formula XIX is a histidine or an asparticacid. Preferably, the amino acid at position 3 of Formula XIX is athreonine or a glutamic acid. Preferably, the amino acid at position 5of Formula XIX is a glutamic acid. Preferably, the amino acid atposition 6 of Formula XIX is an isoleucine, leucine, valine, ortyrosine. Preferably, the amino acid at position 8, 10, 11, or 13 ofFormula XIX is an alanine. Preferably, the amino acid at position 9 ofFormula XIX is an asparagine or a phenylalanine. Preferably, the aminoacid at position 14 of Formula XIX is a glycine.

In further embodiments, GCRA peptides include peptides containing theamino acid sequence of Formula XX. Preferably, the amino acid atposition 1 of Formula XX is a glutamine. Preferably, the amino acid atposition 2 or 3 of Formula XX is a glutamic acid or an aspartic acid.Preferably, the amino acid at position 5 of Formula XX is a glutamicacid. Preferably, the amino acid at position 6 of Formula XX isthreonine, glutamine, tyrosine, isoleucine, or leucine. Preferably, theamino acid at position 8 of Formula XX is isoleucine or valine.Preferably, the amino acid at position 9 of Formula XX is asparagine.Preferably, the amino acid at position 10 of Formula XX is methionine orvaline. Preferably, the amino acid at position 11 of Formula XX isalanine. Preferably, the amino acid at position 13 of Formula XX is athreonione. Preferably, the amino acid at position 1 of Formula XX is aglycine. Preferably, the amino acid at position 15 of Formula XX is atyrosine. Optionally, the amino acid at position 15 of Formula XX is twoamino acid in length and is Cysteine (Cys), Penicillamine (Pen)homocysteine, or 3-mercaptoproline and serine, leucine or threonine.

In certain embodiments, one or more amino acids of the GCC agonistpeptides are replaced by a non-naturally occurring amino acid or anaturally or non-naturally occurring amino acid analog. Such amino acidsand amino acid analogs are known in the art. See, for example, Hunt,“The Non-Protein Amino Acids,” in Chemistry and Biochemistry of theAmino Acids, Barrett, Chapman, and Hall, 1985. In some embodiments, anamino acid is replaced by a naturally-occurring, non-essential aminoacid, e.g., taurine. Non-limiting examples of naturally occurring aminoacids that can be replaced by non-protein amino acids include thefollowing: (1) an aromatic amino acid can be replaced by3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine,L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr); (2) Phg andnorTyr and other amino acids including Phe and Tyr can be substitutedby, e.g., a halogen, —CH3, —OH, —CH2NH3, —C(O)H, —CH2CH3, —CN,—CH2CH2CH3, —SH, or another group; (3) glutamine residues can besubstituted with gamma-Hydroxy-Glu or gamma-Carboxy-Glu; (4) tyrosineresidues can be substituted with an alpha substituted amino acid such asL-alpha-methylphenylalanine or by analogues such as: 3-Amino-Tyr;Tyr(CH3); Tyr(PO3(CH3)2); Tyr(SO3H); beta-Cyclohexyl-Ala;beta-(1-Cyclopentenyl)-Ala; beta-Cyclopentyl-Ala; beta-Cyclopropyl-Ala;beta-Quinolyl-Ala; beta-(2-Thiazolyl)-Ala; beta-(Triazole-1-yl)-Ala;beta-(2-Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe; Fluoro-Phe;Cyclohexyl-Gly; tBu-Gly; beta-(3-benzothienyl)-Ala;beta-(2-thienyl)-Ala; 5-Methyl-Trp; and A-Methyl-Trp; (5) prolineresidues can be substituted with homopro (L-pipecolic acid);hydroxy-Pro; 3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro or anN(alpha)-C(alpha) cyclized amino acid analogues with the structure: n=0,1 , 2, 3; and (6) alanine residues can be substituted withalpha-substituted or N-methylated amino acid such as alpha-aminoisobutyric acid (aib), L/D-alpha-ethylalanine (L/D-isovaline),L/D-methylvaline, or L/D-alpha-methylleucine or a non-natural amino acidsuch as beta-fluoro-Ala. Alanine can also be substituted. with: n=0, 1,2, 3 Glycine residues can be substituted with alpha-amino isobutyricacid (aib) or L/D-alpha- ethylalanine (L/D-isovaline).

Further examples of non-natural amino acids include: an unnatural analogof tyrosine; an unnatural analogue of glutamine; an unnatural analogueof phenylalanine; an unnatural analogue of serine; an unnatural analogueof threonine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine,hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno,ester, thioacid, borate, boronate, phospho, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or aminosubstituted amino acid, or any combination thereof; an amino acid with aphotoactivatable cross-linker; a spin-labeled amino acid; a fluorescentamino acid; an amino acid with a novel functional group; an amino acidthat covalently or noncovalently interacts with another molecule; ametal binding amino acid; an amino acid that is amidated at a site thatis not naturally amidated, a metal-containing amino acid; a radioactiveamino acid; a photocaged and/or photoisomerizable amino acid; a biotinor biotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; amino acidscomprising polyethylene glycol or polyether; a heavy atom substitutedamino acid (e.g., an amino acid containing deuterium, tritium, ¹³C, ¹⁵N,or ¹⁸O); a chemically cleavable or photocleavable amino acid; an aminoacid with an elongated side chain; an amino acid containing a toxicgroup; a sugar substituted amino acid, e.g. , a sugar substituted serineor the like; a carbon-linked sugar-containing amino acid; a redox-activeamino acid; an α-hydroxy containing acid; an amino thio acid containingamino acid; an a, a disubstituted amino acid; a β-amino acid; a cyclicamino acid other than proline; an O-methyl-L-tyrosine; anL-3-(2-naphthyl)alanine; a 3-methyl-phenylalanine; aρ-acetyl-L-phenylalanine; an O-4-allyl-L-tyrosine; a4-propyl-L-tyrosine; a tri-O-acetyl-GlcNAc p-serine; an L-Dopa; afluorinated phenylalanine; an isopropyl-L-phenylalanine; ap-azido-L-phenylalanine; a p-acyl-L-phenylalanine; ap-benzoyl-L-phenylalanine; an L-phosphoserine; a phosphonoserine; aphosphonotyrosine; a p-iodo-phenylalanine; a 4-fluorophenylglycine; ap-bromophenylalanine; a p-amino-L-phenylalanine; anisopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine;D-3-(2-naphthyl)alanine (dNal); an amino-, isopropyl-, orO-allyl-containing phenylalanine analogue; a dopa, O-methyl-L-tyrosine;a glycosylated amino acid; a p-(propargyloxy)phenylalanine;dimethyl-Lysine; hydroxy-proline; mercaptopropionic acid; methyl-lysine;3-nitro-tyrosine; norleucine; pyro-glutamic acid; Z (Carbobenzoxyl);ε-Acetyl-Lysine; β-alanine; aminobenzoyl derivative; aminobutyric acid(Abu); citrulline; aminohexanoic acid; aminoisobutyric acid (AIB);cyclohexylalanine; d-cyclohexylalanine; hydroxyproline; nitro-arginine;nitro-phenylalanine; nitro-tyrosine; norvaline; octahydroindolecarboxylate; ornithine (Orn); penicillamine (PEN);tetrahydroisoquinoline; acetamidomethyl protected amino acids andpegylated amino acids. Further examples of unnatural amino acids andamino acid analogs can be found in U.S. 20030108885, U.S. 20030082575,U.S. PAT. NO. 20060019347 (paragraphs 410-41 8) and the references citedtherein. The polypeptides of the invention can include furthermodifications including those described in U.S. PAT. NO. 20060019347,paragraph 589. Exemplary GCC agonist peptides which include anon-naturally occurring amino acid include for example SP-368 andSP-369.

In some embodiments, the GCC agonist peptides are cyclic peptides. GCCagonist cyclic peptides can be prepared by methods known in the art. Forexample, macrocyclization is often accomplished by forming an amide bondbetween the peptide N- and C-termini, between a side chain and the N- orC-terminus [e.g., with K₃Fe(CN)₅ at pH 8.5] (Samson et al.,Endocrinology, 137: 5182-5185 ( 1996)), or between two amino acid sidechains, such as cysteine. See, e.g., DeGrado, Adv Protein Chem, 39: 51 - 124 ( 1988). In various embodiments, the GCC agonist peptides are[4, 12; 7, 15 ] bicycles.

In certain embodiments, one or both Cys residues which normally form adisulfide bond in a GCC agonist peptide are replaced with homocysteine,penicillamine, 3-mercaptoproline (Kolodziej et al. 1996 Int. J. Pept.Protein Res. 48:274), β, β dimethylcysteine (Hunt et al 1993 Int. J.Pept. Protein Res. 42:249), or diaminopropionic acid (Smith et al. 1978J. Med. Chem. 2 1:117) to form alternative internal cross-links at thepositions of the normal disulfide bonds.

In certain embodiments, one or more disulfide bonds in a GCC agonistpeptide are replaced by alternative covalent cross-links, e.g., an amidelinkage (—CH₂CH(O)NHCH₂— or —CH₂NHCH(O)CH₂—), an ester linkage, athioester linkage, a lactam bridge, a carbamoyl linkage, a urea linkage,a thiourea linkage, a phosphonate ester linkage, an alkyl linkage(—CH₂CH₂CH₂CH₂—), an alkenyl linkage (—CH₂CH═CHCH₂—), an ether linkage(—CH₂CH₂CH₂CH₂— or —CH₂OCH₂CH₂—), a thioether linkage (—CH₂CH₂SCH₂— or—CH₂SCH₂CH₂—), an amine linkage (—CH₂CH₂NHCH₂— or —CH₂NHCH₂CH₂—) or athioamide linkage (—CH₂C(S)NHCH₂— or —CH₂NHC (S)CH₂—). For example, Leduet al. (Proc. Natl. Acad. Sci. 100:11263-78, 2003) describe methods forpreparing lactam and amide cross-links. Exemplary GCC agonist peptideswhich include a lactam bridge include, for example, SP-370.

In certain embodiments, the GCC agonist peptides have one or moreconventional polypeptide bonds replaced by an alternative bond. Suchreplacements can increase the stability of the polypeptide. For example,replacement of the polypeptide bond between a residue amino terminal toan aromatic residue (e.g. Tyr, Phe, Trp) with an alternative bond canreduce cleavage by carboxy peptidases and may increase half-life in thedigestive tract. Bonds that can replace polypeptide bonds include: aretro-inverso bond (C(O)—NH instead of NH—C(O); a reduced amide bond(NH—CH₂); a thiomethylene bond (S—CH₂ or CH₂—S); an oxomethylene bond(O—CH₂ or CH₂—O); an ethylene bond (CH₂—CH₂); a thioamide bond(C(S)—NH); a trans-olefine bond (CH═CH); a fluoro substitutedtrans-olefine bond (CF═CH); a ketomethylene bond (C(O)—CHR or CHR—C(O)wherein R is H or CH₃; and a fluoro-ketomethylene bond (C(O)—CFR orCFR—C(O) wherein R is H or F or CH₃.

In certain embodiments, the GCC agonist peptides are modified usingstandard modifications. Modifications may occur at the amino (N-),carboxy (C-) terminus, internally or a combination of any of thepreceding. In one aspect described herein, there may be more than onetype of modification on the polypeptide. Modifications include but arenot limited to: acetylation, amidation, biotinylation, cinnamoylation,farnesylation, formylation, myristoylation, palmitoylation,phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation,sulfurylation and cyclisation (via disulfide bridges or amidecyclisation), and modification by Cys3 or Cys5. The GCC agonist peptidesdescribed herein may also be modified by 2, 4-dinitrophenyl (DNP),DNP-lysine, modification by 7-Amino-4-methyl- coumarin (AMC),flourescein, NBD (7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide,rhodamine B, EDANS (5-((2-aminoethyl)amino)naphthalene-l-sulfonic acid),dabcyl, dabsyl, dansyl, texas red, FMOC, and Tamra(Tetramethylrhodamine). The GCC agonist peptides described herein mayalso be conjugated to, for example, polyethylene glycol (PEG); alkylgroups (e.g., C1-C20 straight or branched alkyl groups); fatty acidmoieties; combinations of PEG, alkyl groups and fatty acid moieties(See, U.S. Pat. No. 6,309,633; Soltero et al., 2001 Innovations inPharmaceutical Technology 106- 110); BSA and LH (Keyhole LimpetHemocyanin). The addition of PEG and other polymers which can be used tomodify polypeptides of the invention is described in US 20060 19347section IX.

A GCC agonist peptide can also be a derivative of a GCC agonist peptidedescribed herein. For example, a derivative includes hybrid and modifiedforms of GCC agonist peptides in which certain amino acids have beendeleted or replaced. A modification may also include glycosylation.Preferably, where the modification is an amino acid substitution, it isa conservative substitution at one or more positions that are predictedto be non-essential amino acid residues for the biological activity ofthe peptide. A “conservative substitution” is one in which the aminoacid residue is replaced with an amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine. serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

In one embodiment, a GCC agonist peptide prepared by the methodsdescribed herein is subjected to random mutagenesis in order to identifymutants having biological activity.

In one embodiment, the methods of the invention can be used to prepare aGCC agonist peptide that is substantially homologous to a GCC agonistpeptide described herein. Such substantially homologous peptides can beisolated by virtue of cross-reactivity with antibodies to a GCC agonistpeptide described herein.

Further examples of GCC agonist peptides that can be prepared by themethods of the invention are found in Tables I-VII below.

1.1.2 Alternative Preparation Methods of GCC agonist peptides and TheirFragments

GCC agonist peptides and their fragments can be prepared using artrecognized techniques such as molecular cloning, peptide synthesis, orsite-directed mutagenesis.

In addition to the conventional solution- or solid-phase peptidesynthesis described above, the GCC agonist peptides or their fragmentscan be produced by modern cloning techniques. For example, the GCCagonist peptides are produced either in bacteria including, withoutlimitation, E. coli, or in other existing systems for polypeptide orprotein production (e.g., Bacillus subtilis, baculovirus expressionsystems using Drosophila Sf9 cells, yeast or filamentous fungalexpression systems, mammalian cell expression systems), or they can bechemically synthesized. If the GCC agonist peptide or variant peptide isto be produced in bacteria, e.g., E. coli, the nucleic acid moleculeencoding the polypeptide may also encode a leader sequence that permitsthe secretion of the mature polypeptide from the cell. Thus, thesequence encoding the polypeptide can include the pre sequence and thepro sequence of, for example, a naturally-occurring bacterial STpolypeptide. The secreted, mature polypeptide can be purified from theculture medium.

The sequence encoding a GCC agonist peptide described herein can beinserted into a vector capable of delivering and maintaining the nucleicacid molecule in a bacterial cell. The DNA molecule may be inserted intoan autonomously replicating vector (suitable vectors include, forexample, pGE3Z and pcDNA3, and derivatives thereof). The vector nucleicacid may be a bacterial or bacteriophage D A such as bacteriophagelambda or M13 and derivatives thereof. Construction of a vectorcontaining a nucleic acid described herein can be followed bytransformation of a host cell such as a bacterium. Suitable bacterialhosts include but are not limited to, E. coli, B. subtilis, Pseudomonas,Salmonella. The genetic construct also includes, in addition to theencoding nucleic acid molecule, elements that allow expression, such asa promoter and regulatory sequences. The expression vectors may containtranscriptional control sequences that control transcriptionalinitiation, such as promoter, enhancer, operator, and repressorsequences.

A variety of transcriptional control sequences are well known to thosein the art. The expression vector can also include a translationregulatory sequence (e.g., an untranslated 5′ sequence, an untranslated3′ sequence, or an internal ribosome entry site). The vector can becapable of autonomous replication or it can integrate into host DNA toensure stability during polypeptide production.

The protein coding sequence that includes a GCC agonist peptidedescribed herein can also be fused to a nucleic acid encoding apolypeptide affinity tag, e.g., glutathione S-transferase (GST), maltoseE binding protein, protein A, FLAG tag, hexa-histidine, myc tag or theinfluenza HA tag, in order to facilitate purification. The affinity tagor reporter fusion joins the reading frame of the polypeptide ofinterest to the reading frame of the gene encoding the affinity tag suchthat a translational fusion is generated. Expression of the fusion generesults in translation of a single polypeptide that includes both thepolypeptide of interest and the affinity tag. In some instances whereaffinity tags are utilized, DNA sequence encoding a protease recognitionsite will be fused between the reading frames for the affinity tag andthe polypeptide of interest.

Genetic constructs and methods suitable for production of immature andmature forms of the GCC agonist peptides and variants described hereinin protein expression systems other than bacteria, and well known tothose skilled in the art, can also be used to produce polypeptides in abiological system.

The peptides disclosed herein may be modified by attachment of a secondmolecule that confers a desired property upon the peptide, such asincreased half-life in the body, for example, pegylation. Suchmodifications also fall within the scope of the term “variant” as usedherein.

TABLE I GCRA Peptides (SP-304 and Derivatives) Position of SEQ DisulfideID Name bonds Structure NO SP-304 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶1 SP-326 C3: C11, C6: C14Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴-Leu¹⁵2 SP-327 C2: C10, C5: C13Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴3 SP-328 C2: C10, C5: C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Leu¹⁴4 SP-329 C2: C10, C5: C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 5SP-330 C1: C9, C4: C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹²-Leu¹³ 6SP-331 C1: C9, C4: C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹² 7 SP332C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶8 SP-333 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶9 SP-334 C4: C12, C7: C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶10 SP-335 C4: C12, C7: C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶11 SP-336 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶12 SP-337 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-dLeu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶13 SP-338 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵14 SP-342 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG315 SP-343 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG316 SP-344 C4: C12, C7: C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG317 SP-347 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG318 SP-348 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶19 SP-350 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶20 SP-352 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG321 SP-358 C4: C12, C7: C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶⁻22 PEG3 SP-359 C4: C12, C7: C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶23 SP-360 C4: C12, C7: C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶⁻PEG324 SP-361 C4: C12, C7: C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG325 SP-362 C4: C12, C7: C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶26 SP-368 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dNal¹⁶27 SP-369 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-AIB⁸-Asn⁹-AIB¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶28 SP-370 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Asp[Lactam]⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Orn¹⁵-dLeu¹29 SP-371 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶30 SP-372 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶31 N1 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG332 N2 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶33 N3 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶PEG3 34 N4 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG335 N5 C4: C12, C7: C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶36 N6 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG337 N7 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶38 N8 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG339 N9 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶40 N10 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG341 N11 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG342 N12 C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶43 N13 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG344 Formula I C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶45 Formula II C4: C12, C7: C15Xaa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa_(n2)¹⁶ 46 Formula III 4: 12, 7: 15Xaa_(n1)-Maa⁴-Glu⁵-Xaa⁶-Maa⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Maa¹²-Thr¹³-Gly¹⁴-Maa¹⁵-Xaa_(n2)47 Formula IV 4: 12, 7: 15Xaa_(n1)-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaa_(n2)48 Formula V C4: C12, C7: C15Asn¹-Asp²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶49 Formula VI C4: C12, C7: C15dAsn¹-Glu²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-X3⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶50 Formula VII C4: C12, C7: C15dAsn¹-dGlu²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶51 Formula VII C4: C12, C7: C15dAsn¹-dAsp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶52 Formula VIII C4: C12, C7: C15dAsn¹-dAsp²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶53 Formula IX C4: C12, C7: C15dAsn¹-dGlu²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶54

TABLE II Linaclotide and Derivatives Position of SEQ Disulfide ID Namebonds Structure NO: SP-339 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu3-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴55 (linaclotide) SP-340 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 56SP-349 C1: C6, C2: C10, C5: 13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-PEG357 SP-353 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶58 SP-354 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶59 SP-355 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-dTyr¹⁴60 SP-357 C1: C6, C2: C10, C5: 13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴61 SP-374 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶62 SP-375 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶63 SP-376 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶64 SP-377 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶65 SP-378 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶66 SP-379 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶67 SP-380 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶68 SP-381 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶69 SP-382 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶70 SP-383 C3: C8, C4: C12, C7: 15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶71 SP-384 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-PEG372 N14 C1: C6, C2: C10, C5: 13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG373 N15 C1: C6, C2: C10, C5: 13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³74 N16 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG375 N17 C3: C8, C4: C12, C7: 15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-76 Tyr¹⁶-PEG3 N18 C3: C8, C4: C12, C7: 15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-77 Tyr¹⁶ N19 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-78 PEG3 N20 C3: C8, C4: C12, C7: 15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-79 Tyr¹⁶-PEG3 N21 C3: C8, C4: C12, C7: 15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-80 Tyr¹⁶ N22 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-81 PEG3 N23 C3: C8, C4: C12, C7: 15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-82 Tyr¹⁶-PEG3 N24 C3: C8, C4: C12, C7: 15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-83 Tyr¹⁶ N25 C3: C8, C4: C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-84 PEG3 N26 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu3-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴85 N27 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu3-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴86 N28 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu3-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³- 87N29 C1: C6, C2: C10, C5: 13Cys¹-Cys²-Glu3-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 88N30 1: 6, 2: 10, 5: 13Pen¹-Pen²-Glu3-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³-Tyr¹⁴89 N31 1: 6, 2: 10, 5: 13Pen¹-Pen²-Glu3-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³ 90Formula X C9: C14, C10: C18,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Tyr¹²-Cys¹³-Cys¹⁴-Xaa¹⁵-Xaa¹⁶-91 C13: 21 Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XI C9: C14, C10:C18,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Phe⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Phe¹²-Cys¹³-Cys¹⁴-Xaa¹⁵-Xaa¹⁶-92 C13: 21 Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XII C3: C8, C4:C12, C7: 15Asn¹-Phe²-Cys³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷-Cys⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa³-Xaa¹⁴⁻Cys¹⁵-Xaa¹⁶93 Formula XIII 3: 8, 4: 12, C: 15Asn¹-Phe²-Pen³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷-Pen⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys12-Xaa¹³-Xaa¹⁴⁻Cys¹⁵-Xaa¹⁶94 Formula XIV 3: 8, 4: 12, 7: 15Asn¹-Phe²-Maa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Maa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴⁻Maa¹⁵-Xaa¹⁶95 Formula XV 1: 6, 2: 10, 5: 13Maa¹-Maa²-Glu3-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³-Tyr¹⁴96 Formula XVI 1: 6, 2: 10, 5: 13Maa¹-Maa²-Glu3-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³- 97Formula XVII 1: 6, 2: 10, 5: 13Xaa_(n3)-Maa¹-Maa²-Xaa³-Xaa⁴-Maa⁵-Maa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Maa¹⁰-Xaa¹¹-Xaa¹²-Maa¹³-Xaa_(n2)98

TABLE III GCRA Peptides Position of SEQ Disulfide ID Name bondsStructure NO: SP-363 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu-AMIDE¹⁶99 SP-364 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶100 SP-365 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer-AMIDE¹⁶101 SP-366 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶102 SP-367 C4: C12, C7: C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr-AMIDE¹⁶103 SP-373 C4: C12, C7: C15Pyglu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu-AMIDE¹⁶104 SP-304 di C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶-PEG3105 PEG SP-304 N- C4: C12, C7: C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶106 PEG SP-304 C- C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶⁻PEG3107 PEG

TABLE IV SP-304 Analogs, Uroguanylin, and Uroguanylin Analogs Positionof SEQ Disulfide ID Name bonds Structure NO Formula XVIII C4: C12, C7:C15Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaa¹⁶108 Uroguanylin C4: C12, C7: C15Asn¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴⁻Cys¹⁵-Leu¹⁶109 N32 C4: C12, C7: C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶110 N33 C4: C12, C7: C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶111 N34 C4: C12, C7: C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶112 N35 C4: C12, C7: C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶113 N36 C4: C12, C7: C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶114 N37 C4: C12, C7: C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶115 N38 C4: C12, C7: C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶116 N39 C4: C12, C7: C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶117 N40 C4: C12, C7: C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶118 N41 C4: C12, C7: C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶119 N42 C4: C12, C7: C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶120 N43 C4: C12, C7: C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶121 N44 C4: C12, C7: C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶122 N45 C4: C12, C7: C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶123 N46 C4: C12, C7: C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶124 N47 C4: C12, C7: C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶125 N48 C4: C12, C7: C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶126 N49 C4: C12, C7: C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶127 N50 C4: C12, C7: C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶128 N51 C4: C12, C7: C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys-⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶129 N52 C4: C12, C7: C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶130 N53 C4: C12, C7: C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶131 N54 C4: C12, C7: C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶132 N55 C4: C12, C7: C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶133 N56 C4: C12, C7: C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶134 N57 C4: C12, C7: C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶135 N58 C4: C12, C7: C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶136 N59 C4: C12, C7: C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶137 N60 C4: C12, C7: C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶138 N61 C4: C12, C7: C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶139 N62 C4: C12, C7: C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶140 N63 C4: C12, C7: C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶141 N65 C4: C12, C7: C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶142 N66 C4: C12, C7: C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶143 N67 C4: C12, C7: C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶144 N68 C4: C12, C7: C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶145 N69 C4: C12, C7: C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶146 N70 C4: C12, C7: C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶147 N71 C4: C12, C7: C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶148 N72 C4: C12, C7: C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶149 N73 C4: C12, C7: C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶150 N74 C4: C12, C7: C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶151 N75 C4: C12, C7: C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶152 N76 C4: C12, C7: C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶153 N77 C4: C12, C7: C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶154 N78 C4: C12, C7: C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶155 N79 C4: C12, C7: C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶156 N80 C4: C12, C7: C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶157 N81 C4: C12, C7: C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶158 N82 C4: C12, C7: C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶159 N83 C4: C12, C7: C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶160 N84 C4: C12, C7: C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶161 N85 C4: C12, C7: C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶162 N86 C4: C12, C7: C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶163 N87 C4: C12, C7: C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶164 N88 C4: C12, C7: C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶165 N89 C4: C12, C7: C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶166 N90 C4: C12, C7: C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶167 N91 C4: C12, C7: C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶168 N92 C4: C12, C7: C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶169 N93 C4: C12, C7: C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶170 N94 C4: C12, C7: C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶171 N95 C4: C12, C7: C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶172 N96 C4: C12, C7: C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶173

TABLE V Guanylin and Analogs Position of SEQ Disulfide ID Name bondsStructure NO Formula XIX 4: 12, 7: 15Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵174 Guanylin C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Phe⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵175 N97 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵176 N98 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵177 N99 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵178 N100 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵179 N101 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵180 N102 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵181 N103 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵182 N104 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵183 N105 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵184 N106 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵185 N107 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵186 N108 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵187 N109 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵188 N110 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵189 N111 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵190 N112 C4: C12, C7: C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵191 N113 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵192 N114 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵193 N115 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵194 N116 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵195 N117 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵196 N118 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵197 N119 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵198 N120 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵199 N121 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵200 N122 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵201 N123 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵202 N124 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵203 N125 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵204 N126 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵205 N127 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵206 N128 C4: C12, C7: C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵207

TABLE VI Lymphoguanylin and Analogs Position of SEQ Disulfide ID Namebonds Structure NO Formula XX 4: 12, 7: 15Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Xaa_(n1)¹⁵ 208 Lymphoguanylin C4: C12Gln¹-Glu²-Glu-³Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵209 N129 C4: C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵210 N130 C4: C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵211 N131 C4: C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵212 N132 C4: C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵213 N133 C4: C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵214 N134 C4: C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵215 N135 C4: C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵216 N136 C4: C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵217 N137 C4: C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵218 N138 C4: C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵219 N139 C4: C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵220 N140 C4: C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵221 N141 C4: C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵222 N142 C4: C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵223 N143 C4: C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵224 N144 C4: C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵225 N145 C4: C12, C7: C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶226 N146 C4: C12, C7: C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶227 N147 C4: C12, C7: C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶228 N148 C4: C12, C7: C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶229 N149 C4: C12, C7: C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶230 N150 C4: C12, C7: C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser231 N151 C4: C12, C7: C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶232 N152 C4: C12, C7: C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶233 N153 C4: C12, C7: C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶234 N154 C4: C12, C7: C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶235 N155 C4: C12, C7: C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶236 N156 C4: C12, C7: C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶237 N157 C4: C12, C7: C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶238 N158 C4: C12, C7: C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶239 N159 C4: C12, C7: C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶240 N160 C4: C12, C7: C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶241

TABLE VII ST Peptide and Analogues Position of SEQ Disulfide ID Namebonds Structure NO ST C3: C8, C4: C12,Asn¹-Ser²-Ser³-Asn⁴-Ser⁵-Ser⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Glu¹¹-Lys¹²-Cys¹³-Cys¹⁴-Asn¹⁵-Pro¹⁶-Ala¹⁷-Cys¹⁸-242 Peptide C7: 15 Thr¹⁹-Gly²⁰-Cys²¹-Tyr²² N161 C3: C8, C4: C12,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3243 C7: 15 N162 C3: C8, C4: C12,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶244 C7: 15 N163 C3: C8, C4: C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3245 C7: 15 N164 C3: C8, C4: C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶246 C7: 15 N165 C3: C8, C4: C12,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶247 C7: 15 N166 C3: C8, C4: C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶248 C7: 15 N167 C3: C8, C4: C12,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶249 C7: 15

1.2 Methods of Use

The invention provides methods for treating or preventinggastrointestinal disorders and increasing gastrointestinal motility in asubject in need thereof by administering an effective amount of a GCCagonist or a formulation thereof to the subject. Non-limiting examplesof gastrointestinal disorders that can be treated or prevented accordingto the methods of the invention include irritable bowel syndrome (IBS),non-ulcer dyspepsia, H. pylori infection related ulcers, chronicintestinal pseudo-obstruction, functional dyspepsia, colonicpseudo-obstruction, duodenogastric reflux, gastroesophageal refluxdisease (GERD), ileus (e.g., post-operative ileus), gastroparesis,heartburn (high acidity in the GI tract), constipation (e.g.,constipation associated with use of medications such as opioids,osteoarthritis drugs, or osteoporosis drugs); post surgicalconstipation, constipation associated with neuropathic disorders,Crohn's disease, and ulcerative colitis.

In one embodiment, the invention provides methods for treating orpreventing gastrointestinal motility disorder, irritable bowel syndrome,a functional gastrointestinal disorder, gastroesophageal reflux disease,duodenogastric reflux, functional heartburn, dyspepsia, functionaldyspepsia, non-ulcer dyspepsia, gastroparesis, chronic intestinalpseudo-obstruction, colonic pseudo-obstruction, obesity, congestiveheart failure, or benign prostatic hyperplasia.

In one embodiment, the invention provides methods for treating orpreventing constipation and/or increasing gastrointestinal motility in asubject in need thereof by administering an effective amount of a GCCagonist or a formulation thereof to the subject. Clinically acceptedcriteria that define constipation range from the frequency of bowelmovements, the consistency of feces and the ease of bowel movement. Onecommon definition of constipation is less than three bowel movements perweek. Other definitions include abnormally hard stools or defecationthat requires excessive straining (Schiller 2001 Aliment Pharmacol Ther1 5:749-763). Constipation may be idiopathic (functional constipation orslow transit constipation) or secondary to other causes includingneurologic, metabolic or endocrine disorders. These disorders includediabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcaemia,Multiple sclerosis, Parkinson's disease, spinal cord lesions,Neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprungdisease and cystic fibrosis. Constipation may also be the result ofsurgery or due to the use of drugs such as analgesics (like opioids),antihypertensives, anticonvulsants, antidepressants, antispasmodics andantipsychotics.

In various embodiments, the constipation is associated with use of atherapeutic agent; the constipation is associated with a neuropathicdisorder; the constipation is postsurgical constipation; theconstipation is associated with a gastrointestinal disorder; theconstipation is idiopathic (functional constipation or slow transitconstipation); the constipation is associated with neuropathic,metabolic or endocrine disorder (e.g., diabetes mellitus,hypothyroidism, hyperthyroidism, hypocalcaemia, Multiple Sclerosis,Parkinson's disease, spinal cord lesions, neurofibromatosis, autonomicneuropathy, Chagas disease, Hirschsprung disease or cystic fibrosis).Constipation may also be the result of surgery or due to the use ofdrugs such as analgesics (e.g., opioids), antihypertensives,anticonvulsants, antidepressants, antispasmodics and antipsychotics.

In one embodiment, the invention provides methods for treating orpreventing chronic idiopathic constipation and increasinggastrointestinal motility in a subject in need thereof by administeringan effective amount of a GCC agonist or a formulation thereof to thesubject.

The term “treating” as used herein refers to a reduction, a partialimprovement, amelioration, or a mitigation of at least one clinicalsymptom associated with the gastrointestinal disorders being treated.The term “preventing” refers to an inhibition or delay in the onset orprogression of at least one clinical symptom associated with thegastrointestinal disorders to be prevented. The term “effective amount”as used herein refers to an amount that provides some improvement orbenefit to the subject. In certain embodiments, an effective amount isan amount that provides some alleviation, mitigation, and/or decrease inat least one clinical symptom of the gastrointestinal disorder to betreated. In other embodiments, the effective amount is the amount thatprovides some inhibition or delay in the onset or progression of atleast one clinical symptom associated with the gastrointestinal disorderto be prevented. The therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject. The term“subject” preferably refers to a human subject but may also refer to anon-human primate or other mammal preferably selected from among amouse, a rat, a dog, a cat, a cow, a horse, or a pig.

The invention also provides methods for treating gastrointestinal cancerin a subject in need thereof by administering an effective amount of aGCC agonist or a formulation thereof to the subject. Non-limitingexamples of gastrointestinal cancers that can be treated according tothe methods of the invention include gastric cancer, esophageal cancer,pancreatic cancer, colorectal cancer, intestinal cancer, anal cancer,liver cancer, gallbladder cancer, or colon cancer.

The invention also provides methods for treating lipid metabolismdisorders, biliary disorders, inflammatory disorders, lung disorders,cancer, cardiac disorders including cardiovascular disorders, eyedisorders, oral disorders, blood disorders, liver disorders, skindisorders, prostate disorders, endocrine disorders, and obesity.

Lipid metabolism disorders include, but are not limited to,dyslipidemia, hyperlipidemia, hypercholesterolemia,hypertriglyceridemia, sitosterolemia, familial hypercholesterolemia,xanthoma, combined hyperlipidemia, lecithin cholesterol acyltransferasedeficiency, tangier disease, abetalipoproteinemia, erectile dysfunction,fatty liver disease, and hepatitis.

Biliary disorders include gallbladder disorders such as for example,gallstones, gall bladder cancer cholangitis, or primary sclerosingcholangitis; or bile duct disorders such as for example, cholecystitis,bile duct cancer or fascioliasis.

Inflammatory disorders include tissue and organ inflammation such askidney inflammation (e.g., nephritis), gastrointestinal systeminflammation (e.g., Crohn's disease and ulcerative colitis); necrotizingenterocolitis (NEC); pancreatic inflammation (e.g., pancreatis),pancreatic insufficiency, lung inflammation (e.g., bronchitis or asthma)or skin inflammation (e.g., psoriasis, eczema).

Lung Disorders include for example chronic obstructive pulmonary disease(COPD), and fibrosis.

Cancer includes tissue and organ carcinogenesis including metastasessuch as for example gastrointestinal cancer, (e.g., gastric cancer,esophageal cancer, pancreatic cancer colorectal cancer, intestinalcancer, anal cancer, liver cancer, gallbladder cancer, or colon cancer;lung cancer; thyroid cancer; skin cancer (e.g., melanoma); oral cancer;urinary tract cancer (e.g. bladder cancer or kidney cancer); bloodcancer (e.g. myeloma or leukemia) or prostate cancer.

Cardiac disorders include for example, congestive heart failure, tracheacardia hypertension, high cholesterol, or high triglycerides.Cardiovascular disorders include for example aneurysm, angina,atherosclerosis, cerebrovascular accident (stroke),cerebrovasculardisease, congestive heart failure, coronary arterydisease, myocardial infarction (heart attack), or peripheral vasculardisease.

Liver disorders include for example cirrhosis and fibrosis. In addition,GC-C agonist may also be useful to facilitate liver regeneration inliver transplant patients. Eye disorders include for example increasedintra-ocular pressure, glaucoma, dry eyes, retinal degeneration,disorders of tear glands or eye inflammation. Skin disorders include forexample xerosis. Oral disorders include for example dry mouth(xerostomia), Sjögren's syndrome, gum diseases (e.g., periodontaldisease), or salivary gland duct blockage or malfunction. Prostatedisorders include for example benign prostatic hyperplasia (BPH).Endocrine disorders include for example diabetes mellitus,hyperthyroidism, hypothyroidism, and cystic fibrosis.

2.2.1 Therapeutically Effective Dosages

Disorders are treated, prevented or alleviated by administering to asubject, e.g., a mammal such as a human in need thereof, atherapeutically effective dose of a GCC agonist peptide. The presentinvention is based in part on the unexpected results of clinical trialsin humans which demonstrated that the formulations of the invention aretherapeutically effective at much lower doses than predicted based onanimal studies. In accordance with one aspect of the invention, thetherapeutically effective dose is between 0.01 milligrams (mg) and 10 mgper unit dose. The term “unit dose” refers to a single drug deliveryentity, e.g., a tablet, capsule, solution, inhalation, controlledrelease or extended release formulation (e.g. MMX® technology of CosmoPharmaceuticals). In one embodiment, the effective dose is between 0.01mg and 9 mg. In another embodiment, the effective dose is between 0.01mg and 5 mg. In another embodiment, the effective dose is between 0.01mg and 3 mg. In another embodiment, the effective dose is between 0.10mg and 5 mg. In another embodiment, the effective dose is between 0.10mg and 3 mg. In one embodiment, the unit dose is 0.01 mg, 0.05 mg, 0.1mg, 0.2 mg, 0.3 mg, 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 5mg, or 10 mg. In one embodiment, the unit dose is 0.3 mg, 1.0 mg, 3.0mg, 9.0 mg, or 9.5 mg.

The GCC agonist peptides may be in a pharmaceutical composition in unitdose form, together with one or more pharmaceutically acceptableexcipients. The amount of peptide present should be sufficient to have apositive therapeutic effect when administered to a patient. Whatconstitutes a “positive therapeutic effect” will depend upon theparticular condition being treated and will include any significantimprovement in a condition readily recognized by one of skill in theart.

The GCC agonists for use in the methods described above are preferablyadministered orally. Dosage forms include solutions, suspensions,emulsions, tablets, and capsules.

The total daily dose can be administered to the patient in a singledose, or in multiple sub-doses. Typically, sub-doses can be administeredtwo to six times per day, preferably two to four times per day, and evenmore preferably two to three times per day. Preferably, a single dailydose is administered.

The GCC agonists may be administered as either the sole active agent orin combination with one or more additional active agents. In all cases,additional active agents should be administered at a dosage that istherapeutically effective using the existing art as a guide. The GCCagonists may be administered in a single composition or sequentiallywith the one or more additional active agents. In one embodiment, theGCC agonist is administered in combination with one or more inhibitorsof cGMP dependent phosphodiesterase such as suldinac sulfone, zaprinast,motapizone, vardenafil, or sildenifil. In another embodiment, the GCCagonist is administered in combination with one or more chemotherapeuticagents. In another embodiment, the GCC agonist is administered incombination with one or more or anti-inflammatory drugs such as steroidsor non-steroidal anti-inflammatory drugs (NSAIDS), such as aspirin.

Combination therapy can be achieved by administering two or more agents,e.g., a GCC agonist peptide described herein and another compound, eachof which is formulated and administered separately, or by administeringtwo or more agents in a single formulation. Other combinations are alsoencompassed by combination therapy. For example, two agents can beformulated together and administered in conjunction with a separateformulation containing a third agent. While the two or more agents inthe combination therapy can be administered simultaneously, they neednot be. For example, administration of a first agent (or combination ofagents) can precede administration of a second agent (or combination ofagents) by minutes, hours, days, or weeks. Thus, the two or more agentscan be administered within minutes of each other or within 1 , 2, 3, 6,9, 12, 15, 1 8, or 24 hours of each other or within 1 , 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6, 7, 8, 9,or 10 weeks of each other. In some cases even longer intervals arepossible. While in many cases it is desirable that the two or moreagents used in a combination therapy be present in within the patient'sbody at the same time, this need not be so.

The GCC agonist peptides described herein may be combined withphosphodiesterase inhibitors, e.g., sulindae sulfone, Zaprinast,sildenafil, vardenafil or tadalafil to further enhance levels of cGMP inthe target tissues or organs.

Combination therapy can also include two or more administrations of oneor more of the agents used in the combination. For example, if agent Xand agent Y are used in a combination, one could administer themsequentially in any combination one or more times, e.g., in the orderX-Y-X, X-X-Y, Y-X-Y,Y-Y-X,X-X-Y-Y, etc.

1.2.2 Exemplary Agents for Combination Therapy

The GCC agonist formulations of the invention may be administered aloneor in combination with one or more additional therapeutic agents as partof a therapeutic regimen for the treatment or prevention of agastrointestinal disease or disorder. In some embodiments, the GCCagonist formulation comprises one or more additional therapeutic agents.In other embodiments, the GCC agonist is formulated separately from theone or more additional therapeutic agents. In accordance with thisembodiment, the GCC agonist is administered either simultaneously,sequentially, or at a different time than the one or more additionaltherapeutic agents. In one embodiment, the GCC agonist formulation isadministered in combination with one or more additional therapeuticagents selected from the group consisting of phosphodiesteraseinhibitors, cyclic nucleotides (such as cGMP and cAMP), a laxative (suchas SENNA or METAMUCIL), a stool softner, an anti-tumor necrosis factoralpha therapy for IBD (such as REMICADE, ENBREL, or HUMIRA), andanti-inflammatory drugs (such as COX-2 inhibitors, sulfasalazine, 5-ASAderivatives and NSAIDS). In certain embodiments, the GCC agonistformulation is administered in combination with an effective dose of aninhibitor of cGMP-specific phosphodiesterase (cGMP-PDE) eitherconcurrently or sequentially with said GCC agonist. cGMP-PDE inhibitorsinclude, for example, suldinac sulfone, zaprinast, motapizone,vardenifil, and sildenafil. In another embodiment, the GCC agonistformulation is administered in combination with inhibitors of cyclicnucleotide transporters. Further examples of therapeutic agents that maybe administered in combination with the GCC agonist formulations of theinvention are given in the following sections.

1.2.2.1 Agents to Treat Gastrointestinal Cancers

The GCC agonist formulations described herein can be used in combinationwith one or more antitumor agents including but not limited toalkylating agents, epipodophyllotoxins, nitrosoureas, anti-metabolites,vinca alkaloids, anthracycline antibiotics, nitrogen mustard agents, andthe like. Particular antitumor agents include tamoxifen, taxol,etoposide, and 5-fluorouracil. In one embodiment, the GCC agonistformulations are used in combination with an antiviral agent or amonoclonal antibody.

Non-limiting examples of antitumor agents that can be used incombination with the GCC agonist formulations of the invention for thetreatment of colon cancer include anti-proliferative agents, agents forDNA modification or repair, DNA synthesis inhibitors, DNA/RNAtranscription regulators, RNA processing inhibitors, agents that affectprotein expression, synthesis and stability, agents that affect proteinlocalization or their ability to exert their physiological action,agents that interfere with protein-protein or protein-nucleic acidinteractions, agents that act by RNA interference, receptor bindingmolecules of any chemical nature (including small molecules andantibodies), targeted toxins, enzyme activators, enzyme inhibitors, generegulators, HSP-90 inhibitors, molecules interfering with microtubulesor other cytoskeletal components or cell adhesion and motility, agentsfor phototherapy, and therapy adjuncts.

Representative anti-proliferative agents include N-acetyl-D-sphingosine(C.sub.2 ceramide), apigenin, berberine chloride,dichloromethylenediphosphonic acid disodium salt, loe-emodine, emodin,HA 14- 1, N-hexanoyl-D-sphingosine (C.sub.6 ceramide),7b-hydroxycholesterol, 25-hydroxycholesterol, hyperforin, parthenolide,and rapamycin.

Representative agents for DNA modification and repair includeaphidicolin, bleomycin sulfate, carboplatin, carmustine, chlorambucil,cyclophosphamide monohydrate, cyclophosphamide monohydrate ISOPAC.RTM.,cis-diammineplatinum(II) dichloride (Cisplatin), esculetin, melphalan,methoxyamine hydrochloride, mitomycin C, mitoxantrone dihydrochloride,oxaliplatin, and streptozocin.

Representative DNA synthesis inhibitors include (.+−.)amethopterin(methotrexate), 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin,cytosine b-D-arabinofurdnoside (Ara-C), cytosine b-D-arabinofuranoside(Ara-C) hydrochloride, 2-fluoroadenine-9-b-D-arabinofuranoside(Fludarabine des-phosphate; F-ara-A), 5-fluoro-5′-deoxyuridine,5-fluorouracil, ganciclovir, hydroxyurea, 6-mercaptopurine, and6-thioguanine.

Representative DNA/RNA transcription regulators include actinomycin D,daunorubicin hydrochloride, 5,6-dichlorobenzimidazole1-b-D-ribofuranoside, doxorubicin hydrochloride, homoharringtonine, andidarubicin hydrochloride.

Representative enzyme activators and inhibitors include forskolin,DL-aminoglutethimide, apicidin, Bowman-Birk Inhibitor, butein,(S)-(+)-camptothecin, curcumin, (−)-deguelin, (−)-depudecin, doxycyclinehyclate, etoposide, formestane, fostriecin sodium salt, hispidin,2-imino-1-imidazolidineacetic acid (Cyclocreatine), oxamflatin,4-phenylbutyric acid, roscovitine, sodium valproate, trichostatin A,tyrphostin AG 34, tyrphostin AG 879, urinary trypsin inhibitor fragment,valproic acid (2-propylpentanoic acid), and XK469.

Representative gene regulators include 5-aza-2′-deoxycytidine,5-azacytidine, cholecalciferol (Vitamin D3), ciglitizone, cyproteroneacetate, 15-deoxy-D.sup. 12,14-prostaglandin J. sub.2, epitestosterone,flutamide, glycyrrhizic acid ammonium salt (glycyrrhizin),4-hydroxytamoxifen, mifepristone, procainamide hydrochloride, raloxifenehydrochloride, all trans-retinal (vitamin A aldehyde), retinoic acid(vitamin A acid), 9-cis-retinoic acid, 13-cis-retinoic acid, retinoicacid p-hydroxyanilide, retinol (Vitamin A), tamoxifen, tamoxifen citratesalt, tetradecylthioacetic acid, and troglitazone.

Representative HSP-90 inhibitors include17-(allylamino)-17-demethoxygeldanamycin and geldanamycin.

Representative microtubule inhibitors include colchicines, dolastatin15, nocodazole, taxanes and in particular paclitaxel, podophyllotoxin,rhizoxin, vinblastine sulfate salt, vincristine sulfate salt, andvindesine sulfate salt and vinorelbine (Navelbine) ditartrate salt.

Representative agents for performing phototherapy include photoactiveporphyrin rings, hypericin, 5-methoxypsoralen, 8-methoxypsoralen,psoralen and ursodeoxycholic acid.

Representative agents used as therapy adjuncts include amifostine,4-amino-1,8-naphthalimide, brefeldin A, cimetidine, phosphomycindisodium salt, leuprolide (leuprorelin) acetate salt, luteinizinghormone-releasing hormone (LH-RH) acetate salt, lectin, papaverinehydrochloride, pifithrin-a, (−)-scopolamine hydrobromide, andthapsigargin.

The agents can also be anti-VEGF (vascular endothelial growth factor)agents, as such are known in the art. Several antibodies and smallmolecules are currently in clinical trials or have been approved thatfunction by inhibiting VEGF, such as Avastin (Bevacizumab), SU5416,SU11248 and BAY 43-9006. The agents can also be directed against growthfactor receptors such as those of the EGF/Erb-B family such as EGFReceptor (Iressa or Gefitinib, and Tarceva or Erlotinib), Erb-B2,receptor (Herceptin or Trastuzumab), other receptors (such as Rituximabor Rituxan/MabThera), tyrosine kinases, non-receptor tyrosine kinases,cellular serine/threonine kinases (including MAP kinases), and variousother proteins whose deregulation contribute to oncogenesis (such assmall/Ras family and large/heterotrimeric G proteins). Severalantibodies and small molecules targeting those molecules are currentlyat various stages of development (including approved for treatment or inclinical trials).

In a preferred embodiment, the invention provides a method for treatingcolon cancer in a subject in need thereof by-administering to thesubject a GCC agonist formulation in combination with one or moreantitumor agent selected from the group consisting of paclitaxel,docetaxel, tamoxifen, vinorelbine, gemcitabine, cisplatin, etoposide,topotecan, irinotecan, anastrozole, rituximab, trastuzumab, fludarabine,cyclophosphamide, gentuzumab, carboplatin, interferons, and doxorubicin.In a particular embodiment the antitumor agent is paclitaxel. In afurther embodiment, the method further comprises an antitumor agentselected from the group consisting of 5-FU, doxorubicin, vinorelbine,Cytoxan, and cisplatin.

1.2.2.2 Agents that Treat Crohn's Disease

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of Crohn's disease.Non-limiting examples of the one or more additional therapeutic agentsinclude sulfasalazine and other mesalamine-containing drugs, generallyknown as 5-ASA agents, such as Asacol, Dipentum, or Pentasa, orinfliximab (REMICADE). In certain embodiments, the one or moreadditional agents is a corticosteroid or an immunosuppressive agent suchas 6-mercaptopurine or azathioprine. In another embodiment, the one ormore additional agents is an antidiarrheal agent such as diphenoxylate,loperamide, or codeine.

1.2.2.3 Agents that Treat Ulcerative Colitis

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of ulcerative colitis.The agents that are used to treat ulcerative colitis overlap with thoseused to treat Chrohn's Disease. Non-limiting examples of the one or moreadditional therapeutic agents that can be used in combination with a GCCagonist formulation of the invention include aminosalicylates (drugsthat contain 5-aminosalicyclic acid (5-ASA)) such as sulfasalazine,olsalazine, mesalamine, and balsalazide. Other therapeutic agents thatcan be used include corticosteroids, such as prednisone andhydrocortisone, immunomodulators, such as azathioprine,6-mercapto-purine (6-MP), cytokines, interleukins, and lymphokines, andanti-TNF-alpha agents, including the thiazolidinediones or glitazonessuch as rosiglitazone and pioglitazone. In one embodiment, the one ormore additional therapeutic agents includes both cyclosporine A and 6-MPor azathioprine for the treatment of active, severe ulcerative colitis.

1.2.2.4 Agents that Treat Constipation/Irritable Bowel Syndrome

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of constipation, such asthat associated with irritable bowel syndrome. Non-limiting examples ofthe one or more additional therapeutic agents include laxatives such asSENNA, MIRALAX, LACTULOSE, PEG, or calcium polycarbophil), stoolsofteners (such as mineral oil or COLACE), bulking agents (such asMETAMUCIL or bran), agents such as ZELNORM (also called tegaserod), andanticholinergic medications such as BENTYL and LEVSIN.

1.2.2.5 Agents for the Treatment of Postoperative Ileus

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of postoperative ileus.Non-limiting examples of the one or more additional therapeutic agentsinclude ENTEREG (alvimopan; formerly called ado lor/ADL 8-2698),conivaptan, and related agents describes in U.S. Pat. No. 6,645,959.

1.2.2.6 Anti-Obesity Agents

In one embodiment, a GCC agonist formulation of the invention isadministered as part of a combination therapy with one or moreadditional therapeutic agents for the treatment of obesity. Non-limitingexamples of the one or more additional therapeutic agents include 1 βHSD-1 (1-beta hydroxy steroid dehydrogenase type 1) inhibitors, such asBVT 3498, BVT 2733, 3-(1-adamantyl)-4-ethyl-5-(ethylthio)-4H-1,2,4-triazole,3-(1-adamantyl)-5-(3,4,5-trimethoxyphenyl)-4-methyl-4H-1,2,4-triazole,3-adamantanyl-4,5,6,7,8,9,10,11,12,3a-decahydro-1,2,4-triazolo[4,3-a][11]annulene,and those compounds disclosed in WO01/90091, WOO 1/90090, WOO 1/90092and WO02/072084; 5HT antagonists such as those in WO03/037871 ,WO03/037887, and the like; 5HTIa modulators such as carbidopa,benserazide and those disclosed in U.S. Pat. No. 6,207,699, WO03/031439, and the like; 5HT2c (serotonin receptor 2c) agonists, such asBVT933, DPCA37215, IK264, PNU 22394, WAY161503, R-1065, SB 243213 (GlaxoSmith Kline) and YM 348 and those disclosed in U.S. Pat. No. 3,914,250,WO00/77010, WO02/36596, WO02/48124, WO02/10169, WO01/66548, WO02/44152,WO02/51844, WO02/40456, and WO02/40457; 5HT6 receptor modulators, suchas those in WO03/030901, WO03/035061 , WO03/039547, and the like;acyl-estrogens, such as oleoyl-estrone, disclosed in del Mar-Grasa, M.et al, Obesity Research, 9:202-9 (2001 ) and Japanese Patent ApplicationNo. JP 2000256190; anorectic bicyclic compounds such as 1426 (Aventis)and 1954 (Aventis), and the compounds disclosed in WO00/18749,WO01/32638, WO01/62746, WO01/62747, and WO03/015769; CB 1 (cannabinoid-1receptor) antagonist/inverse agonists such as rimonabant (Acomplia;Sanofi), SR- 147778 (Sanofi), SR- 141 716 (Sanofi), BAY 65-2520 (Bayer),and SLV 319 (Solvay), and those disclosed in patent publications U.S.Pat. No. 4,973,587, U.S. Pat. No. 5,013,837, U.S. Pat. No. 5,081,122,U.S. Pat. No. 5,112,820, U.S. Pat. No. 5,292,736, U.S. Pat. No.5,532,237, U.S. Pat. No. 5,624,941 , U.S. Pat. No. 6,028,084, U.S. Pat.No. 6,509,367, U.S. Pat. No. 6,509,367, WO96/33159, WO97/29079,WO98/31227, WO98/33765, WO98/37061 , WO98/41519, WO98/43635, WO98/43636,WO99/02499, WO00/10967, WO00/10968, WO01/09120, WO01 /58869, WO01/64632, WO01/64633, WO01/64634, WO01/70700, WO01/96330, WO02/076949,WO03/006007, WO03/007887, WO03/020217, WO03/026647, WO03/026648,WO03/027069, WO03/027076, WO03/0271 14, WO03/037332, WO03/040107,WO03/086940, WO03/084943 and EP658546; CC -A (cholecystokinin-A)agonists, such as AR-R 15849, GI 181771 (GSK), JMV-180, A-71378, A-71623and SR 146131 (Sanofi), and those described in us5,739,106; CNTF(Ciliary neurotrophic factors), such as GI-181771 (Glaxo-SmithKline),SRI 46131 (Sanofi Synthelabo), butabindide, PD 170,292, and PD 149164(Pfizer); CNTF derivatives, such as Axokine® (Regeneron), and thosedisclosed in WO94/09134, WO98/22128, and WO99/43813; dipeptidylpeptidase IV (DP-IV) inhibitors, such as isoleucine thiazolidide, valinepyrrolidide, NVP-DPP728, LAF237, P93/01, P 3298, TSL 225(tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; disclosedby Yamada et al, Bioorg. & Med. Chem. Lett. 8 (1998) 1537-1540),TMC-2A/2B/2C, CD26 inhibtors, FE 999011, P9310/K364, VIP 0177, SDZ274-444, 2-cyanopyrrolidides and 4-cyanopyrrolidides as disclosed byAshworth et al, Bioorg. & Med. Chem. Lett., Vol. 6, No. 22, pp 1163-1166and 2745-2748 (1996) and the compounds disclosed patent publications.WO99/38501 , WO99/46272, WO99/67279 (Probiodrug), W099/67278(Probiodrug), W099/61431 (Probiodrug), WO02/083128, WO02/062764,WO03/0001 80, WO03/000181 , WO03/000250, WO03/002530, WO03/002531,WO03/002553, WO03/002593, WO03/004498, WO03/004496,WO03/017936,WO03/024942, WO03/024965, WO03/033524, WO03/037327 and EP 1 258476;growth hormone secretagogue receptor agonists/antagonists, such asNN703, hexarelin, MK-0677 (Merck), SM-130686, CP-424391 (Pfizer), LY444,711 (Eli Lilly), L-692,429 and L-163,255, and such as thosedisclosed in U.S. PAT. NO. SN 09/662448, U.S. provisional application60/203335, U.S. Pat. No. 6,358,951 , U.S. PAT. NO. 2002049196, U.S. PAT.NO. 2002/022637, WO01 /56592 and WO02/32888; H3 (histamine H3)antagonist/inverse agonists, such as thioperamide, 3-(IH-imidazol-4-yl)propyl N-(4-pentenyl)carbamate), clobenpropit, iodophenpropit,imoproxifan, GT2394 (Gliatech), and A331440,O-[3-(1H-imidazol-4-yl)propanol]carbamates (Kiec-Kononowicz, K. et al.,Pharmazie, 55:349-55 (2000)), piperidine-containing histamineH3-receptor antagonists (Lazewska, D. et al., Pharmazie, 56:927-32 (2001), benzophenone derivatives and related compounds (Sasse, A. et al.,Arch. Pharm.(Weinheim) 334:45-52 (2001 )), substituted N-phenylcarbamates (Reidemeister, S. et al., Pharmazie, 55:83-6 (2000)),and proxifan derivatives (Sasse, A. et al., J. Med. Chem. 43:3335-43(2000)) and histamine H3 receptor modulators such as those disclosed inWO02/15905, WO03/024928 and WO03/024929; leptin derivatives, such asthose disclosed in U.S. Pat. No. 5,552,524, us5,552,523, U.S. Pat. No.5,552,522, U.S. Pat. No. 5,521,283, WO96/23513, WO96/23514, WO96/23515,WO96/23516, WO96/23517, WO96/23518, WO96/23519, and WO96/23520; leptin,including recombinant human leptin (PEG-OB, Hoffman La Roche) andrecombinant methionyl human leptin (Amgen); lipase inhibitors, such astetrahydrolipstatin (orlistat/Xenical®), Triton WR1339, RHC80267,lipstatin, teasaponin, diethylumbelliferyl phosphate, FL-386,WAY-121898, Bay-N-3 176, valilactone, esteracin, ebelactone A,ebelactone B, and RHC 80267, and those disclosed in patent publivcationsWO01/77094, U.S. Pat. No. 4,598,089, U.S. Pat. No. 4,452,813, U.S. Pat.No. 5,512,565, U.S. Pat. No. 5,391,571, U.S. Pat. No. 5,602,151 , U.S.Pat. No. 4,405,644, U.S. Pat. No. 4,189,438, and U.S. Pat. No.4,242,453; lipid metabolism modulators such as maslinic acid,erythrodiol, ursolic acid uvaol, betulinic acid, betulin, and the likeand compounds disclosed in WO03/01, 1267; Mc4r (melanocortin 4 receptor)agonists, such as CHIR86036 (Chiron), ME-10142, ME-10145, and HS-131(Melacure), and those disclosed in PCT publication Nos. WO99/64002,WO00/74679, WO01/991752, WOO 1/25192, WOO 1/52880, WOO 1/74844, WOO1/70708, WO01/70337, WO01/91752, WO02/059095, WO02/059107, WO02/059108,WO02/059117, WO02/06276, WO02/12166, WO02/11715, WO02/12178, WO02/15909,WO02/38544, WO02/068387, WO02/068388, WO02/067869, WO02/081430,WO03/06604, WO03/007949, WO03/009847, WO03/009850, WO03/013509, andWO03/031410; Mc5r (melanocortin 5 receptor) modulators, such as thosedisclosed in WO97/19952, WO00/15826, WO00/15790, U.S. PAT. NO.20030092041 ; melanin-concentrating hormone 1 receptor (MCHR)antagonists, such as T-226296 (Takeda), SB 568849, SNP-7941 (Synaptic),and those disclosed in patent publications WOO 1/21169, WO01/82925,WO01/87834, WO02/051809, WO02/06245, WO02/076929, WO02/076947,WO02/04433, WO02/51809, WO02/083134, WO02/094799, WO03/004027,WO03/13574, WO03/15769, WO03/028641, WO03/035624, WO03/033476,WO03/033480, JP 13226269, and JP 1437059; mGluR5 modulators such asthose disclosed in WO03/029210, WO03/047581, WO03/048137, WO03/051315,WO03/051833, WO03/053922, WO03/059904, and the like; serotoninergicagents, such as fenfluramine (such as Pondimin® (Benzeneethanamine,N-ethyl-alpha-methyl-3-(trifluoromethyl)-, hydrochloride), Robbins),dexfenfluramine (such as Redux® (Benzeneethanamine,N-ethyl-alpha-methyI-3-(trifluoromethyl)-, hydrochloride), Interneuron)and sibutramine ((Meridia®, Knoll/Reductil™) including racemic mixtures,as optically pure isomers (+) and (−), and pharmaceutically acceptablesalts, solvents, hydrates, clathrates and prodrugs thereof includingsibutramine hydrochloride monohydrate salts thereof, and those compoundsdisclosed in U.S. Pat. No. 4,746,680, U.S. Pat. No. 4,806,570, and U.S.Pat. No. 5,436,272, U.S. Pat. No. 20020006964, WOO 1/27068, and WOO1/62341 ; NE (norepinephrine) transport inhibitors, such as GW 320659,despiramine, talsupram, and nomifensine; NPY 1 antagonists, such asBIBP3226, J-115814, BIBO 3304, LY-357897, CP-671906, GI-264879A, andthose disclosed in U.S. Pat. No. 6,001,836, WO96/14307, WO01/23387,WO99/51600, WO01/85690, WO01/85098, WO01/85173, and WO01/89528; NPY5(neuropeptide Y Y5) antagonists, such as 152,804, GW-569180A,GW-594884A, GW- 587081X, GW-548118X, FR235208, FR226928, FR240662,FR252384, 12291U91, GI-264879A, CGP71683A, LY-377897, LY-366377,PD-160170, SR-120562A, SR-120819A, JCF-104, and H409/22 and thosecompounds disclosed in patent publications U.S. Pat. No. 6,140,354, U.S.Pat. No. 6,191,160, U.S. Pat. No. 6,218,408, U.S. Pat. No. 6,258,837,U.S. Pat. No. 6,313,298, U.S. Pat. No. 6,326,375, U.S. Pat. No.6,329,395, U.S. Pat. No. 6,335,345, U.S. Pat. No. 6,337,332, U.S. Pat.No. 6,329,395, U.S. Pat. No. 6,340,683, EP01 010691 , EP-01044970,WO97/19682, WO97/20820, WO97/20821 , WO97/20822, WO97/20823, WO98/27063,WO00/107409, WO00/1 85714, WO00/185730, WO00/64880, WO00/68197,WO00/69849, WO/0113917, WO01 /09120, WO01/14376, WO01 /85714,WO01/85730, WO01/07409, WO01/02379, WO01/23388, WO01/23389, WOO1/44201,WO01/62737, WO01/62738, WO01/09120, WO02/20488, WO02/22592, WO02/48152,WO02/49648, WO02/051806, WO02/094789, WO03/009845, WO03/014083,WO03/022849, WO03/028726 and Norman et al., J. Med. Chem. 43:4288-4312(2000); opioid antagonists, such as nalmefene (REVEX®),3-methoxynaltrexone, methylnaltrexone, naloxone, and naltrexone (e.g.PT901; Pain Therapeutics, Inc.) and those disclosed in U.S. PAT. NO.20050004155 and WO00/21 509; orexin antagonists, such as SB-334867-A andthose disclosed in patent publications WOO1/96302, WOO1/68609,WO02/44172, WO02/51232, WO02/5 1 838, WO02/089800, WO02/090355,WO03/023561, WO03/032991, and WO03/037847; PDE inhibitors (e.g.compounds which slow the degradation of cyclic AMP (cAMP) and/or cyclicGMP (cGMP) by inhibition of the phosphodiesterases, which can lead to arelative increase in the intracellular concentration of cAMP and cGMP;possible PDE inhibitors are primarily those substances which are to benumbered among the class consisting of the PDE3 inhibitors, the classconsisting of the PDE4 inhibitors and/or the class consisting of thePDE5 inhibitors, in particular those substances which can be designatedas mixed types of PDE3/4 inhibitors or as mixed types of PDE3/4/5inhibitors) such as those disclosed in patent publications DE 1470341 ,DE2108438, DE2123328, DE2305339, DE2305575, DE2315801, DE2402908,DE2413935, DE2451417, DE2459090, DE2646469, DE2727481, DE2825048,DE2837161 , DE2845220, DE2847621 , DE2934747, DE3021792, DE3038166,DE3044568, EP00071 8, EP0008408, EP0010759, EP0059948, EP0075436,EP0096517, EPO1 12987, EPO1 16948, EP01 50937, EP0158380, EP0161632,EP0161918, EP0167121 , EP0199127, EP0220044, EP0247725, EP0258191 ,EP0272910, EP0272914, EP0294647, EP0300726, EP0335386, EP0357788,EP0389282, EP0406958, EP0426180, EP0428302, EP0435811, EP0470805,EP0482208, EP0490823, EP0506194, EP0511865, EP0527117, EP0626939,EP0664289, EP0671389, EP0685474, EP0685475, EP0685479, JP92234389,JP94329652, JP95010875, U.S. Pat. No. 4,963,561, U.S. Pat. No. 5,141,931, WO9117991, WO9200968, WO9212961, WO9307146, WO9315044, WO9315045,WO9318024, WO9319068, WO9319720, WO9319747, WO9319749, WO9319751,WO9325517, WO9402465, WO9406423, WO9412461, WO9420455, WO9422852,WO9425437, WO9427947, WO9500516, WO9501980, WO9503794, WO9504045,WO9504046, WO9505386, WO9508534, WO9509623, WO9509624, WO9509627,WO9509836, WO9514667, WO9514680, WO9514681, WO9517392, WO9517399,WO9519362, WO9522520, WO9524381, WO9527692, WO9528926, WO9535281,WO9535282, WO9600218, WO9601825, WO9602541, WO9611917, DE3142982,DE1116676, DE2162096, EP0293063, EP0463756, EP0482208, EP0579496,EP0667345 U.S. Pat. No. 6,331,543, U.S. Pat. No. 20050004222 (includingthose disclosed in formulas I-XIII and paragraphs 37-39, 85-0545 and557-577), WO9307124, EP0163965, EP0393500, EP0510562, EP0553174,WO9501338 and WO9603399, as well as PDE5 inhibitors (such as RX-RA-69,SCH-51866, KT-734, vesnarinone, zaprinast, SKF-96231 , ER-21355,BF/GP-385, NM-702 and sildenafil (Viagra™)), PDE4 inhibitors (such asetazolate, ICI63197, RP73401, imazolidinone (RO-20-1724), MEM 1414(R1533/R1500; Pharmacia Roche), denbufylline, rolipram, oxagrelate,nitraquazone, Y-590, DH-6471, SKF-94120, motapizone, lixazinone,indolidan, olprinone, atizoram, KS-506-G, dipamfylline, BMY-43351 ,atizoram, arofylline, filaminast, PDB-093, UCB-29646, CDP-840,SKF-107806, piclamilast, RS-17597, RS-25344-000, SB-207499, TIBENELAST,SB-210667, SB-211572, SB-211600, SB-212066, SB-212179, GW-3600, CDP-840,mopidamol, anagrelide, ibudilast, amrinone, pimobendan, cilostazol,quazinone andN-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide,PDE3 inhibitors (such as 1C11 53, 100, bemorandane (RWJ 22867), MCI-154,UD-CG 212, sulmazole, ampizone, cilostamide, carbazeran, piroximone,imazodan, CI-930, siguazodan, adibendan, saterinone, S F-95654, SDZ-MS-492, 349-U-85, emoradan, EMD-53998, EMD-57033, NSP-306, NSP-307,revizinone, NM-702, W1N-62582 and WIN-63291 , enoximone and milrinone,PDE3/4 inhibitors (such as benafentrine, trequinsin, ORG-30029,zardaverine, L-686398, SDZ-1SQ-844, ORG-20241 , EMD-54622, andtolafentrine) and other PDE inhibitors (such as vinpocetin, papaverine,enprofylline, cilomilast, fenoximone, pentoxifylline, roflumilast,tadalafil(Cialis®), theophylline, and vardenafil(Levitra©); NeuropeptideY2 (NPY2) agonists include but are not limited to: polypeptide YY andfragments and variants thereof (e.g. YY3-36 (PYY3-36 )(N. Engl. J. Med.349:941, 2003; IKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY (SEQ ID NO:XXX))and PYY agonists such as those disclosed in WO02/47712, WO03/026591,WO03/057235, and WO03/027637; serotonin reuptake inhibitors, such as,paroxetine, fluoxetine (Prozac™), fluvoxamine, sertraline, citalopram,and imipramine, and those disclosed in U.S. Pat. No. 6,162,805, U.S.Pat. No. 6,365,633, WO03/00663, WOO 1/27060, and WOO 1/162341; thyroidhormone β agonists, such as KB-261 1 (KaroBioBMS), and those disclosedin WO02/15845, WO97/21993, WO99/00353, GB98/284425, U.S. ProvisionalApplication No. 60/183,223, and Japanese Patent Application No. JP2000256190; UCP-I (uncoupling protein-1), 2, or 3 activators, such asphytanic acid, 4-[(E)-2-(5, 6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-i-propenyl]benzoic acid(TTNPB), retinoic acid, and those disclosed in WO99/00123; β3 (betaadrenergic receptor 3) agonists, such as AJ9677/TAK677(Dainippon/Takeda), L750355 (Merck), CP331648 (Pfizer), CL-316,243, SB418790, BRL-37344, L-796568, BMS-196085, BRL-35135A, CGP12177A, BTA-243,GW 427353, Trecadrine, Zeneca D7114, N-5984 (Nisshin Kyorin), LY-377604(Lilly), SR 59119A, and those disclosed in U.S. Pat. No. 5,541,204, U.S.Pat. No. 5,770,615, U.S. Pat. No. 5,491,134, U.S. Pat. No. 5,776,983,U.S. Pat. No. 488,064, U.S. Pat. No. 5,705,515, us5,451,677, WO94/18161,WO95/29159, WO97/46556, WO98/04526 and WO98/32753, WO01/74782,WO02/32897, WO03/014113, WO03/016276, WO03/016307, WO03/024948,WO03/024953 and WO03/037881 ; noradrenergic agents including, but notlimited to, diethylpropion (such as Tenuate® (1-propanone,2-(diethylamino)-1-phenyl-, hydrochloride), Merrell), dextroamphetamine(also known as dextroamphetamine sulfate, dexamphetamine, dexedrine,Dexampex, Ferndex, Oxydess II, Robese, Spancap #1 ), mazindol ((or5-(p-chlorophenyl)-2,5-dihydro-3H- imidazo[2,1-a]isoindol-5-ol) such asSanorex®, Novartis or Mazanor®, Wyeth Ayerst), phenylpropanolamine (orBenzenemethanol, alpha-(1-aminoethyl)-, hydrochloride), phentermine ((orPhenol, 3-[[4,5-duhydro-IH-imidazol-2-yl)ethyl](4-methylpheny-1)amino],monohydrochloride) such as Adipex-P®, Lemmon, FASTIN®, Smith-KlineBeecham and lonamin®, Medeva), phendimetrazine ((or(2S,3S)-3,4-Dimethyl-2phenylmorpholine L-(+)-tartrate (1:1)) such asMetra® (Forest), Plegine® (Wyeth-Ayerst), Prelu-2® (BoehringerIngelheim), and Statobex® (Lemmon), phendamine tartrate (such asThephorin® (2,3,4,9-Tetrahydro-2-methyl-9-phenyl-1H-indenol[2,1-c]pyridine L-(+)-tartrate(1:1)), Hoffmann-LaRoche), methamphetamine (such as Desoxyn®, Abbot((S)-N, (alpha)-dimethylbenzeneethanamine hydrochloride)), andphendimetrazine tartrate (such as Bontril® Slow-Release Capsules, Amarin(-3,4-Dimethyl-2-phenylmorpholine Tartrate); fatty acid oxidationupregulator/inducers such as Famoxin® (Genset); monamine oxidaseinhibitors including but not limited to befloxatone, moclobemide,brofaromine, phenoxathine, esuprone, befol, toloxatone, pirlindol,amiflamine, sercloremine, bazinaprine, lazabemide, milacemide,caroxazone and other certain compounds as disclosed by WOO 1 /12176; andother anti-obesity agents such as 5HT-2 agonists, ACC (acetyl-CoAcarboxylase) inhibitors such as those described in WO03/072197,alpha-lipoic acid (alpha-LA), AOD9604, appetite suppressants such asthose in WO03/40107, ATL-962 (Alizyme PLC), benzocaine, benzphetaminehydrochloride (Didrex), bladderwrack (focus vesiculosus), BRS3 (bombesinreceptor subtype 3) agonists, bupropion, caffeine, CCK agonists,chitosan, chromium, conjugated linoleic acid, corticotropin-releasinghormone agonists, dehydroepiandrosterone, DGAT1 (diacylglycerolacyltransferase 1) inhibitors, DGAT2 (diacylglycerol acyltransferase 2)inhibitors, dicarboxylate transporter inhibitors, ephedra, exendin-4 (aninhibitor of glp-1) FAS (fatty acid synthase) inhibitors (such asCerulenin and C75), fat resorption inhibitors (such as those inWO03/053451, and the like), fatty acid transporter inhibitors, naturalwater soluble fibers (such as psyllium, plantago, guar, oat, pectin),galanin antagonists, galega (Goat's Rue, French Lilac), garciniacambogia, germander (teucrium chamaedrys), ghrelin antibodies andghrelin antagonists (such as those disclosed in WO01/87335, andWO02/08250), polypeptide hormones and variants thereof which affect theislet cell secretion, such as the hormones of the secretin/gastricinhibitory polypeptide (GIP)/vasoactive intestinal polypeptide(VIP)/pituitary adenylate cyclase activating polypeptide(PACAP)/gIucagon-like polypeptide II (GLP-II)/glicentin/gIucagon genefamily and/or those of the adrenomedullin/amylin/calcitonin gene relatedpolypeptide (CGRP) gene family including GLP-1 (glucagon-likepolypeptide 1 ) agonists (e.g. (1) exendin-4, (2) those GLP-I moleculesdescribed in U.S. PAT. NO. 20050130891 including GLP-1 (7-34),GLP-1(7-35), GLP-1(7-36) or GLP-1(7-37) in its C-terminally carboxylatedor amidated form or as modified GLP-1 polypeptides and modificationsthereof including those described in paragraphs 17-44 of U.S. PAT. NO.20050130891, and derivatives derived from GLP-1-(7-34)COOH and thecorresponding acid amide are employed which have the following generalformula: R-NH-HAEGTFTSDVSYLEGQAAEFIAWLVK-CONH₂ wherein R═H or an organiccompound having from 1 to 10 carbon atoms. Preferably, R is the residueof a carboxylic acid. Particularly preferred are the followingcarboxylic acid residues: formyl, acetyl, propionyl, isopropionyl,methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl.) andglp-1 (glucagon-like polypeptide-1), glucocorticoid antagonists, glucosetransporter inhibitors, growth hormone secretagogues (such as thosedisclosed and specifically described in U.S. Pat. No. 5,536,716),interleukin-6 (IL-6) and modulators thereof (as in WO03/057237, and thelike), L-carnitine, Mc3r (melanocortin 3 receptor) agonists, MCH2R(melanin concentrating hormone 2R) agonist/antagonists, melaninconcentrating hormone antagonists, melanocortin agonists (such asMelanotan II or those described in WO 99/64002 and WO 00/74679), nomameherba, phosphate transporter inhibitors, phytopharm compound 57 (CP644,673), pyruvate, SCD-I (stearoyl-CoA desaturase-1) inhibitors, T71(Tularik, Inc., Boulder Colo.), Topiramate (Topimax®, indicated as ananti-convulsant which has been shown to increase weight loss),transcription factor modulators (such as those disclosed inWO03/026576), β-hydroxy steroid dehydrogenase-1 inhibitors (β-HSD-I),β-hydroxy-β-methylbutyrate, p57 (Pfizer), Zonisamide (Zonegran™,indicated as an anti-epileptic which has been shown to lead to weightloss), and the agents disclosed in US20030119428 paragraphs 20-26.

1.2.2.7 Phosphodiesterase inhibitors

In certain embodiments, the regimen of combination therapy includes theadministration of one or more phosphodiesterase (“PDE”) inhibitors. PDEinhibitors slow the degradation of cyclic AMP (cAMP) and/or cyclic GMP(cGMP) by inhibiting phosphodiesterases, which can lead to a relativeincrease in the intracellular concentration of cAMP and/or cGMP.Non-limiting examples of PDE inhibitors that can be used in combinationwith the GCC agonists of the invention include PDE3 inhibitors, PDE4inhibitors and/or PDE5 inhibitors, in particular those substances whichcan be designated as mixed types of PDE3/4 inhibitors or as mixed typesof PDE3/4/5 inhibitors. Non-limiting examples of such PDE inhibitors aredescribed in the following patent applications and patents: DE1470341,DE2108438, DE2123328, DE2305339, DE2305575, DE2315801, DE2402908,DE2413935, DE2451417, DE2459090, DE2646469, DE2727481, DE2825048,DE2837161, DE2845220, DE2847621, DE2934747, DE3021792, DE3038166,DE3044568, EP000718, EP0008408, EP0010759, EP0059948, EP0075436,EP0096517, EPO112987, EPO116948, EP0150937, EP0158380, EP0161632,EP0161918, EP0167121, EP0199127, EP0220044, EP0247725, EP0258191,EP0272910, EP0272914, EP0294647, EP0300726, EP0335386, EP0357788,EP0389282, EP0406958, EP0426180, EP0428302, EP0435811, EP0470805,EP0482208, EP0490823, EP0506194, EP0511865, EP0527117, EP0626939,EP0664289, EP0671389, EP0685474, EP0685475, EP0685479, JP92234389,JP94329652, JP95010875, U.S. Pat. Nos. 4,963,561, 5,141,931, WO9117991,WO9200968, WO9212961, WO9307146, WO9315044, WO9315045, WO9318024,WO9319068, WO9319720, WO9319747, WO9319749, WO9319751, WO9325517,WO9402465, WO9406423, W09412461 , WO9420455, WO9422852, WO9425437,WO9427947, WO9500516, WO9501980, WO9503794, WO9504045, WO9504046,WO9505386, WO9508534, WO9509623, WO9509624, WO9509627, WO9509836,WO9514667, WO9514680, WO9514681, WO09517392, WO09517399, WO9519362,WO9522520, WO9524381, WO9527692, WO9528926, WO9535281, WO9535282,WO9600218, WO9601825, WO9602541, WO9611917, DE3142982, DE1116676,DE2162096, EP0293063, EP0463756, EP0482208, EP0579496, EP0667345 U.S.Pat. No. 6,331,543, US20050004222 (including those disclosed in formulasI-XIII and paragraphs 37-39, 85-0545 and 557-577) and WO9307124,EPO163965, EP0393500, EP0510562, EP0553174, WO9501338 and WO9603399.PDE5 inhibitors which may be mentioned by way of example are RX-RA-69,SCH-51866, KT-734, vesnarinone, zap inast, SKF-96231, ER-21355,BF/GP-385, NM-702 and sildenafil (Viagra®). PDE4 inhibitors which may bementioned by way of example are RO-20-1724, MEM 1414 (R1533/R1500;Pharmacia Roche), DENBUFYLLINE, ROLIPRAM, OXAGRELATE, NITRAQUAZONE,Y-590, DH-6471, SKF-94120, MOTAPIZONE, LIXAZINONE, INDOLIDAN, OLPRINONE,ATIZORAM, KS-506-G, DIPAMFYLLINE, BMY-43351, ATIZORAM, AROFYLLINE,FILAMINAST, PDB-093, UCB-29646, CDP-840, SKF-107806, PICLAMILAST,RS-17597, RS-25344-000, SB-207499, TIBENELAST, SB-210667, SB-211572,SB-211600, SB-212066, SB-212179, GW-3600, CDP-840, MOPIDAMOL,ANAGRELIDE, IBUDILAST, AMRINONE, PIMOBENDAN, CILOSTAZOL, QUAZINONE andN-(3,5-dichloropyrid-4-yl)-3-cyclopropylmethoxy4-difluoromethoxybenzamide.PDE3 inhibitors which may be mentioned by way of example are SULMAZOLE,AMPIZONE, CILOSTAMIDE, CARBAZERAN, PIROXIMONE, IMAZODAN, CI-930,SIGUAZODAN, ADIBENDAN, SATERINONE, SKF-95654, SDZ-MKS-492, 349-U-85,EMORADAN, EMD-53998, EMD-57033, NSP-306, NSP-307, REVIZINONE, NM-702,WIN-62582 and WIN-63291, ENOXIMONE and MILRINONE. PDE3/4 inhibitorswhich may be mentioned by way of example are BENAFENTRINE, TREQUINSIN,ORG-30029, ZARDAVERINE, L-686398, SDZ-ISQ-844, ORG-20241, EMD-54622, andTOLAFENTRINE. Other PDE inhibitors include: cilomilast, pentoxifylline,roflumilast, tadalafil(Cialis®), theophylline, and vardenafil(Levitra®),zaprinast (PDE5 specific). GCC AGONIST

1.2.2.8 Analgesic Agents

In certain embodiments, the regimen of combination therapy includes theadministration of one or more analgesic agents, e.g., an analgesiccompound or an analgesic polypeptide. In some embodiments, the GCCagonist formulation is administered simultaneously or sequentially withone or more analgesic agents. In other embodiments, the GCC agonist iscovalently linked or attached to an analgesic agent to create atherapeutic conjugate. Non-limiting examples of analgesic agents thatcan be used include calcium channel blockers, 5HT receptor antagonists(for example 5HT3, 5HT4 and 5HT1 receptor antagonists), opioid receptoragonists (loperamide, fedotozine, and fentanyl), NK1 receptorantagonists, CC receptor agonists (e.g., loxiglumide), NK1 receptorantagonists, NK3 receptor antagonists, norepinephrine-serotonin reuptakeinhibitors (NSRI), vanilloid and cannabanoid receptor agonists, andsialorphin. Further examples of analgesic agents in the various classesare known in the art.

In one embodiment, the analgesic agent is an analgesic polypeptideselected from the group consisting of sialorphin-related polypeptides,including those comprising the amino acid sequence QHNPR (SEQ ID NO:239), including: VQHNPR (SEQ ID NO: 240); VRQHNPR (SEQ ID NO: 241);VRGQHNPR (SEQ ID NO: 242); VRGPQHNPR (SEQ ID NO: 243); VRGPRQHNPR (SEQID NO: 244); VRGPRRQHNPR (SEQ ID NO: 245); and RQHNPR (SEQ ID NO: 246).Sialorphin-related polypeptides bind to neprilysin and inhibitneprilysin-mediated breakdown of substance P and Met-enkephalin. Thus,compounds or polypeptides that are inhibitors of neprilysin are usefulanalgesic agents which can be administered with the GCC agonistsdescribed herein or covalently linked to a GCC agonist to form atherapeutic conjugate. Sialorphin and related polypeptides are describedin U.S. Pat. No. 6,589,750; U.S. 20030078200 A1; and WO 02/051435 A2.

In another embodiment, a GCC agonist formulation of the invention isadministered as part of a regimen of combination therapy with an opioidreceptor antagonist or agonist. In one embodiment, the GCC agonist andthe opioid receptor antagonist or agonist are linked via a covalentbond. Non-limiting examples of opioid receptor antagonists includenaloxone, naltrexone, methyl nalozone, nalmefene, cypridime, betafunaltrexamine, naloxonazine, naltrindole, nor-binaltorphimine,enkephalin pentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine),trimebutine, vasoactive intestinal polypeptide, gastrin, glucagons.Non-limiting examples of opioid receptor agonists include fedotozine,asimadoline, and ketocyclazocine, the compounds described in WO03/097051and WO05/007626, morphine, diphenyloxylate, frakefamide(H-Tyr-D-Ala-Phe(F)-Phe-NH 2; WO 01/019849 A1), and loperamide.

Further non-limiting examples of analgesic agents that can be used in aregimen of combination therapy along with the GCC agonist formulationsof the invention include the dipeptide Tyr-Arg (kyotorphin); thechromogranin-derived polypeptide (CgA 47-66; See, e.g., Ghia et al. 2004Regulatory polypeptides 119:199); CCK receptor agonists such ascaerulein; conotoxin polypeptides; peptide analogs of thymulin (FRApplication 2830451); CCK (CCKa or CCKb) receptor antagonists, includingloxiglumide and dexloxiglumide (the R-isomer of loxiglumide) (WO88/05774); 5-HT4 agonists such as tegaserod (Zelnorm®), mosapride,metoclopramide, zacopride, cisapride, renzapride, benzimidazolonederivatives such as BIMU 1 and BIMU 8, and lirexapride; calcium channelblockers such as ziconotide and related compounds described in, forexample, EP625162B1 , U.S. Pat. No. 5,364,842, U.S. Pat. No. 5,587,454,U.S. Pat. No. 5,824,645, U.S. Pat. No. 5,859,186, U.S. Pat. No.5,994,305, U.S. Pat. No. 6,087,091 , U.S. Pat. No. 6,136,786, WO93/13128 A1, EP 1336409 A1, EP 835126 A1, EP 835126 B1, U.S. Pat. No.5,795,864, U.S. Pat. No. 5,891,849, U.S. Pat. No. 6,054,429, WO 97/01351A1; NK-1, receptor antagonists such as aprepitant (Merck & Co Inc),vofopitant, ezlopitant (Pfizer, Inc.), R-673 (Hoffmann-La Roche Ltd),SR-48968 (Sanofi Synthelabo), CP-122,721 (Pfizer, Inc.), GW679769 (GlaxoSmith Kline), TAK-637 (Takeda Abbot), SR-14033, and related compoundsdescribed in, for example, EP 873753 A1, U.S. Pat. No. 20010006972 A1,U.S. Pat. No. 20030109417 A1, WO 01/52844 A1 (for a review see Giardinaet al. 2003.Drugs 6:758); NK-2 receptor antagonists such as nepadutant(Menarini Ricerche SpA), saredutant (Sanofi-Synthelabo), GW597599 (GlaxoSmith Kline), SR-1441 90 (Sanofi-Synthelabo) and UK-290795 (Pfizer Inc);NK3 receptor antagonists such as osanetant (SR-142801;Sanofi-Synthelabo), SSR-241586, talnetant and related compoundsdescribed in, for example, WO 02/094187 A2, EP 876347 A1, WO 97/21680A1, U.S. Pat. No. 6,277,862, WO 98/11090, WO 95/28418, WO 97/19927, andBoden et al. (J Med Chem. 39: 1664-75, 1996); norepinephrine-serotoninreuptake inhibitors (NSRI) such as milnacipran and related compoundsdescribed in WO 03/077897; and vanilloid receptor antagonists such asarvanil and related compounds described in WO 01/64212 A1.

In addition to sialorphin-related polypeptides, analgesic polypeptidesinclude: AspPhe, endomorphin-1 , endomorphin-2, nocistatin, dalargin,lupron, ziconotide, and substance P.

1.2.2.9 Insulin and Insulin Modulating Agents

The GCC agonist peptides described herein can be used in combinationtherapy with insulin and related compounds including primate, rodent, orrabbit insulin including biologically active variants thereof includingallelic variants, more preferably human insulin available in recombinantform. Sources of human insulin include pharmaceutically acceptable andsterile formulations such as those available from Eli Lilly(Indianapolis, Ind. 46285) as Humulin™ (human insulin rDNA origin). See,the THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed. (2001) MedicalEconomics, Thomson Healthcare (disclosing other suitable humaninsulins).

The GCC peptides described herein can also be used in combinationtherapy with agents that can boost insulin effects or levels of asubject upon administration, e.g. glipizide and/or rosiglitazone. Thepolypeptides and agonists described herein can be used in combitherapywith SYMLIN® (pramlintide acetate) and Exenatide®(synthetic exendin-4; a39 aa polypeptide).

1.2.2.10 Anti-Hypertensive Agents

The GCC agonist peptides described herein can be used in combinationtherapy with an anti-hypertensive agent including but not limited to:(1) diuretics, such as thiazides, including chlorthalidone,chlorthiazide, dichlorophenamide, hydroflumethiazide, indapamide,polythiazide, and hydrochlorothiazide; loop diuretics, such asbumetanide, ethacrynic acid, furosemide, and torsemide; potassiumsparing agents, such as amiloride, and triamterene; carbonic anhydraseinhibitors, osmotics(such as glycerin) and aldosterone antagonists, suchas spironolactone, epirenone, and the like; (2) beta-adrenergic blockerssuch as acebutolol, atenolol, betaxolol, bevantolol, bisoprolol,bopindolol, carteolol, carvedilol, celiprolol, esmolol, indenolol,metaprolol, nadolol, nebivolol, penbutolol, pindolol, propanolol,sotalol, tertatolol, tilisolol, and timolol, and the like; (3) calciumchannel blockers such as amlodipine, aranidipine, azelnidipine,barnidipine, benidipine, bepridil, cinaldipine, clevidipine, diltiazem,efonidipine, felodipine, gallopamil, isradipine, lacidipine,lemildipine, lercanidipine, nicardipine, nifedipine, nilvadipine,nimodepine, nisoldipine, nitrendipine, manidipine, pranidipine, andverapamil, and the like; (4) angiotensin converting enzyme (ACE)inhibitors such as benazepril; captopril; ceranapril; cilazapril;delapril; enalapril; enalopril; fosinopril; imidapril; lisinopril;losinopril; moexipril; quinapril; quinaprilat; ramipril; perindopril;perindropril; quanipril; spirapril; tenocapril; trandolapril, andzofenopril, and the like; (5) neutral endopeptidase inhibitors such asomapatrilat, cadoxatril and ecadotril, fosidotril, sampatrilat, AVE7688,ER4030, and the like; (6) endothelin antagonists such as tezosentan,A308165, and YM62899, and the like; (7) vasodilators such ashydralazine, clonidine, minoxidil, and nicotinyl alcohol, and the like;(8) angiotensin II receptor antagonists such as aprosartan, candesartan,eprosartan, irbesartan, losartan, olmesartan, pratosartan, tasosartan,telmisartan, valsartan, and EXP-3137, FI6828K, and RNH6270, and thelike; (9) α/β adrenergic blockers such as nipradilol, arotinolol andamosulalol, and the like; (10) alpha 1 blockers, such as terazosin,urapidil, prazosin, tamsulosin, bunazosin, trimazosin, doxazosin,naftopidil, indoramin, WHP 164, and XENOIO, and the like; (11) alpha 2agonists such as lofexidine, tiamenidine, moxonidine, rilmenidine andguanobenz, and the like; (12) aldosterone inhibitors, and the like; and(13) angiopoietin-2-binding agents such as those disclosed inWO03/030833. Specific anti-hypertensive agents that can be used incombination with polypeptides and agonists described herein include, butare not limited to: diuretics, such as thiazides (e.g., chlorthalidone,cyclothiazide (CAS RN 2259-96-3), chlorothiazide (CAS RN 72956-09-3,which may be prepared as disclosed in U.S. Pat. No. 2,809,194),dichlorophenamide, hydroflumethiazide, indapamide, polythiazide,bendroflumethazide, methyclothazide, polythiazide, trichlormethazide,chlorthalidone, indapamide, metolazone, quinethazone, althiazide (CASRIM 5588-16-9, which may be prepared as disclosed in British Patent No.902,658), benzthiazide (CAS RN 91-33-8, which may be prepared asdisclosed in us3,108,097), buthiazide (which may be prepared asdisclosed in British Patent Nos. 861,367), and hydrochlorothiazide),loop diuretics (e.g. bumetanide, ethacrynic acid, furosemide, andtorasemide), potassium sparing agents (e.g. amiloride, and triamterene(CAS Number 396-01-O)), and aldosterone antagonists (e.g. spironolactone(CAS Number 52-01-7), epirenone, and the like); β-adrenergic blockerssuch as Amiodarone (Cordarone, Pacerone), bunolol hydrochloride (CAS RN31969-05-8, Parke-Davis), acebutolol(±N-[3-Acetyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl]-butanamide,or (±)-3′-Acetyl-4′-[2-hydroxy-3-(isopropylamino) propoxy]butyranilide),acebutolol hydrochloride (e.g. Sectral®, Wyeth-Ayerst), alprenololhydrochloride (CAS RN 13707-88-5 see Netherlands Patent Application No.6,605,692), atenolol (e.g. Tenormin®, AstraZeneca), carteololhydrochloride (e.g. Cartrol® Filmtab®, Abbott), Celiprolol hydrochloride(CAS RN 57470-78-7, also see in U.S. Pat. No. 4,034,009), cetamololhydrochloride (CAS RN 77590-95-5, see also U.S. Pat. No. 4,059,622),labetalol hydrochloride (e.g. Normodyne®, Schering), esmololhydrochloride (e.g. Brevibloc®, Baxter), levobetaxolol hydrochloride(e.g. Betaxon™ Ophthalmic Suspension, Alcon), levobunolol hydrochloride(e.g. Betagan® Liquifilm® with C CAP® Compliance Cap, Allergan), nadolol(e.g. Nadolol, Mylan), practolol (CAS RN 6673-35-4, see also U.S. Pat.No. 3,408,387), propranolol hydrochloride (CAS RN 318-98-9), sotalolhydrochloride (e.g. Betapace AF™,Berlex), timolol (2-Propanol, 1-[(1,1-dimethylethyl)amino]-3-[[4-4(4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy]-,hemihydrate, (S)—, CAS RN 91524-16-2), timolol maleate(S)-1-[(1,1-dimethylethyl)amino]-3-[[4-(4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy]-2-propanol(Z)-2-butenedioate (1:1) salt, CAS RN 26921-17-5), bisoprolol(2-Propanol,1-[4-[[2-(1-methylethoxy)ethoxy]-methyl]phenoxyl]-3-[(1-meth-ylethyl)amino]-,(±), CAS RN 66722-44-9), bisoprolol fumarate (such as(±)-1-[4-[[2-(1-ethylethoxy)ethoxy]methyl]phenoxy]-3-[(1-methylethyl)amino]-2-propanol(E)-2-butenedioate (2:1) (salt), e.g., Zebeta™, Lederle Consumer),nebivalol (2H-1-Benzopyran-2-methanol,αα′-[iminobis(methylene)]bis[6-fluoro-3,4-dihydro-, CAS RN 99200-09-6see also U.S. Pat. No. 4,654,362), cicloprolol hydrochloride, such2-Propanol,1-[4-[2-(cyclopropylmethoxy)ethoxy]phenoxy]-3-[1-methylethyl)amino]-,hydrochloride, A.A.S. RN 63686-79-3), dexpropranolol hydrochloride(2-Propanol,1-[1-methylethy)-amino]-3-(1-naphthalenyloxy)-hydrochloride(CAS RN 13071-11-9), diacetolol hydrochloride (Acetamide,N-[3-acetyl-4-[2-hydroxy-3-[(1-methyl-ethyl)amino]propoxy][phenyl]-,monohydrochloride CAS RN 69796-04-9), dilevalol hydrochloride(Benzamide,2-hydroxy-5-[1-hydroxy-2-[1-methyl-3-phenylpropyl)amino]methyl]-,monohydrochloride, CAS RN 75659-08-4), exaprolol hydrochloride(2-Propanol, 1-(2-cyclohexylphenoxy)-3-[(1-methylethyl)amino]-,hydrochloride CAS RN 59333-90-3), flestolol sulfate (Benzoic acid,2-fluro-,3-[[2-[aminocarbonyl)amino]-dimethylethyl]amino]-2-hydroxypropyl ester,(+)-sulfate (1:1) (salt), CAS RN 88844-73-9; metalol hydrochloride(Methanesulfonamide, N-[4-[1-hydroxy-2-(methylamino)propyl]phenyl]-,monohydrochloride CAS RN 7701-65-7), metoprolol 2-Propanol, 1-[4-(2-methoxyethyl)phenoxy]-3-[1-methylethyl)amino]-; CAS RN 37350-58-6),metoprolol tartrate (such as 2-Propanol,1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)amino]-, e.g.,Lopressor®, Novartis), pamatolol sulfate (Carbamic acid,[2-[4-[2-hydroxy-3-[(1-methylethyl)amino]propoxyl]phenyl]-ethyl]-,methyl ester, (±) sulfate (salt) (2:1), CAS RN 59954-01-7), penbutololsulfate (2-Propanol, 1-(2-cyclopentylphenoxy)-3-[1,1dimethyle-thyl)amino]1, (S)-, sulfate (2:1) (salt), CAS RN 38363-32-5),practolol (Acetamide,N-[4-[2-hydroxy-3-[(1-methylethyl)amino]-propoxy]phenyl]-, CAS RN6673-35-4;) tiprenolol hydrochloride (Propanol,1-[(1-methylethyl)amino]-3-[2-(methylthio)-phenoxy]-, hydrochloride,(±), CAS RN 39832-43-4), tolamolol (Benzamide,4-[2-[[2-hydroxy-3-(2-methylphenoxy)-propyl]amino]ethoxyl]-, CAS RN38103-61-6), bopindolol, indenolol, pindolol, propanolol, tertatolol,and tilisolol, and the like; calcium channel blockers such as besylatesalt of amlodipine (such as3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylatebenzenesulphonate, e.g., Norvasc®, Pfizer), clentiazem maleate(1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-8-chloro-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2-(4-methoxyphenyl)-(2S-cis)-,(Z)-2-butenedioate (1:1), see also U.S. Pat. No. 4,567,195), isradipine(3,5- Pyridinedicarboxylic acid,4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-, methyl 1-methylethylester, (±)-4(4-benzofurazanyl)-1 ,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate, see also U.S. Pat. No. s4,466,972); nimodipine(such as is isopropyl (2- methoxyethyl) 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5- pyridine-dicarboxylate, e.g. Nimotop®, Bayer),felodipine (such as ethyl methyl4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate-, e.g. Plendil® Extended-Release, AstraZeneca LP), nilvadipine(3,5-Pyridinedicarboxylic acid,2-cyano-1,4-dihydro-6-methyl-4-(3-nitrophenyl)-,3-methyl5-(1-methylethyl) ester, also see U.S. Pat. No. 3,799,934), nifedipine(such as 3,5-pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester, e.g., ProcardiaXL® Extended Release Tablets, Pfizer), diltiazem hydrochloride (such as1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-dihydro-2(4-methoxyphenyl)-,monohydrochloride, (+)-cis., e.g., Tiazac®, Forest), verapamilhydrochloride (such as benzeneacetronitrile,(alpha)-[[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-(alpha)-(1-methylethyl)hydrochloride, e.g., Isoptin® SR, Knoll Labs), teludipine hydrochloride(3,5-Pyridinedicarboxylic acid,2-[(dimethylamino)methyl]4-[2-[(1E)-3-(1,1-dimethylethoxy)-3-oxo-1-propenyl]phenyl]-1,4-dihydro-6-methyl-,diethyl ester, monohydrochloride) CAS RN 108700-03-4), belfosdil(Phosphonic acid, [2-(2-phenoxy ethyl)-1,3-propane-diyl]bis-, tetrabutylester CAS RN 103486-79-9), fostedil (Phosphonic acid,[[4-(2-benzothiazolyl)phenyl]methyl]-, diethyl ester CAS RN 75889-62-2),aranidipine, azelnidipine, barnidipine, benidipine, bepridil,cinaldipine, clevidipine, efonidipine, gallopamil, lacidipine,lemildipine, lercanidipine, monatepil maleate (1-Piperazinebutanamide,N-(6, 11-dihydrodibenzo(b,e)thiepin-11-yl)4-(4-fluorophenyl)-, (+)-,(Z)-2-butenedioate (1:1) (±)-N-(6,1 1-Dihydrodibenzo(b,e)thiep-in-11-yl)-4-(p-fluorophenyl)-1-piperazinebutyramide maleate (1:1) CAS RN132046-06-1), nicardipine, nisoldipine, nitrendipine, manidipine,pranidipine, and the like; T-channel calcium antagonists such asmibefradil; angiotensin converting enzyme (ACE) inhibitors such asbenazepril, benazepril hydrochloride (such as 3-[[1-(ethoxycarbonyl)-3-phenyl-(1S)-propyl]amino]-2,3,4,5-tetrahydro-2-oxo-1H-1-(3S)-benzazepine-1-aceticacid monohydrochloride, e.g., Lotrel®, Novartis), captopril (such as1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline, e.g., Captopril, Mylan,CAS RN 62571-86-2 and others disclosed in U.S. Pat. No. 4,046,889),ceranapril (and others disclosed in U.S. Pat. No. 4,452,790), cetapril(alacepril, Dainippon disclosed in Eur. Therap. Res. 39:671 (1986);40:543 ( 1986)), cilazapril (Hoffman-LaRoche) disclosed in J.Cardiovasc. Pharmacol. 9:39 (1987), indalapril (delapril hydrochloride(2H-1,2,4-Benzothiadiazine-7-sulfonamide, 3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-, 1,1-dioxide CAS RN 2259-96-3);disclosed in U.S. Pat. No. 4,385,051 ), enalapril (and others disclosedin U.S. Pat. No. 4,374,829), enalopril, enaloprilat, fosinopril, ((suchas L-proline, 4-cyclohexyl-1-[[[2-methyl-1-(1-oxopropoxy)propoxy](4-phenylbutyl) phosphinyl]acetyl]-, sodium salt, e.g.,Monopril, Bristol-Myers Squibb and others disclosed in U.S. Pat. No.4,168,267), fosinopril sodium (L-Proline,4-cyclohexyl-1-[[(R)-[(1S)-2-methyl-1-(1-ox-opropoxy)propox), imidapril,indolapril (Schering, disclosed in J. Cardiovasc. Pharmacol. 5:643, 655(1983)), lisinopril (Merck), losinopril, moexipril, moexiprilhydrochloride (3-Isoquinolinecarboxylic acid,2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]-1,-2,3,4-tetrahydro-6,7-dimethoxy-,monohydrochloride, (3S)-CAS RN 82586-52-5), quinapril, quinaprilat,ramipril (Hoechsst) disclosed in EP 79022 and Curr. Ther. Res. 40:74(1986), perindopril erbumine (such as2S,3aS,7aS-1-[(S)-N-[(S)-1-Carboxybutyl]alanyl]hexahydro-indolinecarboxylic acid, 1-ethyl ester, compound with tert-butylamine(1:1), e.g., Aceon®, Solvay), perindopril (Servier, disclosed in Eur. J.clin. Pharmacol. 31:519 (1987)), quanipril (disclosed in U.S. Pat. No.4,344,949), spirapril (Schering, disclosed in Acta. Pharmacol. Toxicol.59 (Supp. 5): 173 (1986)), tenocapril, trandolapril, zofenopril (andothers disclosed in U.S. Pat. No. 4,316,906), rentiapril (fentiapril,disclosed in Clin. Exp. Pharmacol. Physiol. 10:131 (1983)), pivopril,YS980, teprotide (Bradykinin potentiator BPP9a CAS RN 35115-60-7), BRL36,378 (Smith Kline Beecham, see EP80822 and EP60668), MC-838 (Chugai,see CA. 102:72588v and Jap. J. Pharmacol. 40:373 (1986), CGS 14824(Ciba-Geigy,3-([1-ethoxycarbonyl-3-phenyl-(1S)-propyl]amino)-2,3,4,5-tetrahydro-2-ox-o-1-(3S)-benzazepine-1acetic acid HCl, see U.K. Patent No. 2103614), CGS 16,617 (Ciba-Geigy,3(S)-[[(1S)-5-amino-1-carboxypentyl]amino]-2,3,4,-5-tetrahydro-2-oxo-1H-1- benzazepine-1-ethanoic acid, see us4,473,575),Ru 44570 (Hoechst, see Arzneimittelforschung 34:1254 (1985)), R31-2201(Hoffman-LaRoche see FEBS Lett. 165:201 (1984)), C1925 (Pharmacologist26:243, 266 (1984)), WY-44221 (Wyeth, see J. Med. Chem. 26:394 (1983)),and those disclosed in U.S. Pat. No. 2003006922 (paragraph 28), U.S.Pat. No. 4,337,201, U.S. Pat. No. 4,432,971 (phosphonamidates); neutralendopeptidase inhibitors such as omapatrilat (Vanev®), CGS 30440,cadoxatril and ecadotril, fasidotril (also known as aladotril oralatriopril), sampatrilat, mixanpril, and gemopatrilat, AVE7688, ER4030,and those disclosed in U.S. Pat. No. 5,362,727, U.S. Pat. No. 5,366,973,U.S. Pat. No. 5,225,401 , U.S. Pat. No. 4,722,810, U.S. Pat. No.5,223,516, U.S. Pat. No. 4,749,688, U.S. Pat. No. 5,552,397, U.S. Pat.No. 5,504,080, U.S. Pat. No. 5,612,359, U.S. Pat. No. 5,525,723,EP0599444, EP0481522, EP0599444, EP0595610, EP0534363, EP534396,EP534492, EP0629627; endothel in antagonists such as tezosentan,A308165, and YM62899, and the like; vasodilators such as hydralazine(apresoline), clonidine (clonidine hydrochloride (1H-Imidazol-2-amine,N-(2,6-dichlorophenyl)4,5-dihydro-, monohydrochloride CAS RN 4205-91-8),catapres, minoxidil (loniten), nicotinyl alcohol (roniacol), diltiazemhydrochloride (such as1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-dihydro-2(4-methoxyphenyl)-,monohydrochloride, (+)-cis, e.g., Tiazac®, Forest), isosorbide dinitrate(such as 1,4:3,6-dianhydro-D-glucitol 2,5-dinitrate e.g., Isordil®Titradose®, Wyeth-Ayerst), sosorbide mononitrate (such as1,4:3,6-dianhydro-D-glucito-1,5-nitrate, an organic nitrate, e.g.,Ismo®, Wyeth-Ayerst), nitroglycerin (such as 2,3 propanetrioltrinitrate, e.g., Nitrostat® Parke-Davis), verapamil hydrochloride (suchas benzeneacetonitrile, (±)-(alpha)[3-[[2-(3,4 dimethoxypheny1)ethyl]methylamino]propyl]-3,4-dimethoxy-(alpha)-(1-methylethyl)hydrochloride, e.g., Covera HS® Extended-Release, Searle), chromonar(which may be prepared as disclosed in U.S. Pat. No. 3,282,938),clonitate (Annalen 1870 155), droprenilamine (which may be prepared asdisclosed in DE2521113), lidoflazine (which may be prepared as disclosedin U.S. Pat. No. 3,267,104); prenylamine (which may be prepared asdisclosed in U.S. Pat. No. 3,152,173), propatyl nitrate (which may beprepared as disclosed in French Patent No. 1,103,113), mioflazinehydrochloride (1-Piperazineacetamide,3-(aminocarbonyl)4-[4,4-bis(4-fluorophenyl)butyl]-N-(2,6-dichlorophenyl)-, dihydrochloride CAS RN 83898-67-3), mixidine(Benzeneethanamine,3,4-dimethoxy-N-(1-methyl-2-pyrrolidinylidene)-Pyrrolidine,2-[(3,4-dimethoxyphenethyl)imino]-1-methyl-1-Methyl-2-[(3,4-dimethoxyphenethyl)imino]pyrrolidineCAS RN 27737-38-8), molsidomine (1,2,3-Oxadiazolium,5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt CAS RN25717-80-0), isosorbide mononitrate (D-Glucitol, 1,4:3,6-dianhydro-,5-nitrate CAS RN 16051-77-7), erythrityl tetranitrate(1,2,3,4-Butanetetrol, tetranitrate, (2R,3S)-rel-CAS RN 7297-25-8),clonitrate(1,2-Propanediol, 3-chloro-, dinitrate (7Cl, 8Cl, 9Cl) CAS RN2612-33-1), dipyridamole Ethanol,2,2′,2″,2″′-[(4,8-di-1-piperidinylpyrimido[5,4-d]pyrimidine-2,6-diyl)dinitrilo]tetrakis-CASRN 58-32-2), nicorandil (CAS RN 65141-46-0 3-), pyridinecarboxamide(N-[2-(nitrooxy)ethyl]-Nisoldipine3,5-Pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, methyl 2-methylpropyl esterCAS RN 63675-72-9), nifedipines, 5-Pyridinedicarboxylic acid,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester CAS RN21829-25-4), perhexiline maleate (Piperidine,2-(2,2-dicyclohexylethyl)-, (2Z)-2-butenedioate (1:1) CAS RN 6724-53-4),oxprenolol hydrochloride (2-Propanol,1-[(1-methylethyl)amino]-3-[2-(2-propenyloxy)phenoxy]-, hydrochlorideCAS RN 6452-73-9), pentrinitrol (1,3-Propanediol,2,2-bis[(nitrooxy)methyl]-, mononitrate (ester) CAS RN 1607-17-6),verapamil (Benzeneacetonitrile, α-83-[[2-(3,4-dimethoxyphenyl)ethyl]-methylamino]propyl]-3,4-dimethoxy-α-(1-methylethyl)-CAS RN 52-53-9) and the like; angiotensin II receptor antagonists suchas, aprosartan, zolasartan, olmesartan, pratosartan, FI6828K, RNH6270,candesartan (1 H-Benzimidazole-7-carboxylic acid,2-ethoxy-1[[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]4-yl]methyl]-CAS RN139481-59-7), candesartan cilexetil((+/−)-1-(cyclohexylcarbonyloxy)ethyl-2-ethoxy-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]-1H-benzimidazolecarboxylate, CAS RN 145040-37-5, U.S. Pat. No. 5,703,110 and U.S. Pat.No. 5,196,444), eprosartan(3-[1-4-carboxyphenylmethyl)-2-n-butyl-imidazol-5-yl]-(2-thienylmethyl)propenoic acid, U.S. Pat. No. s5,185,351 and U.S. Pat. No. 5,650,650),irbesartan (2-n-butyl-3-[[2′-(1h-tetrazol-5-yl)biphenyl-4-yl]methyl]1,3-diazazspiro[4,4]non-1-en-4-one, U.S. Pat. No. 5,270,317 and U.S.Pat. No. 5,352,788), losartan(2-N-butyl-4-chloro-5-hydroxymethyl-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole,potassium salt, U.S. Pat. No. 5,138,069, U.S. Pat. No. 5,153,197 andU.S. Pat. No. 5,128,355), tasosartan(5,8-dihydro-2,4-dimethyl-8-[(2′-(1H-tetrazol-5-yl)[1,r-biphenyl]4-yl)methyl]-pyrido[2,3-d]pyrimidin-7(6H)-one,U.S. Pat. No. 5,149,699), telmisartan(4′-[(1,4-dimethyl-2′-propyl-(2,6′-bi-1H-benzimidazol)-r-yl)]-[1,1′-biphenyl]-2-carboxylic acid, CAS RN 144701-48-4, U.S. Pat. No.5,591,762), milfasartan, abitesartan, valsartan (Diovan® (Novartis),(S)-N-valeryl-N-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]valine, U.S.Pat. No. 5,399,578), EXP-3137(2-N-butyl-4-chloro-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole-5-carboxylicacid, U.S. Pat. No. 5,138,069, U.S. Pat. No. 5,153,197 and U.S. Pat. No.5,128,355),3-(2′-(tetrazol-5-yl)-1,r-biphen-4-yl)methyl-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine,4′[2-ethyl-4-methyl-6-(5,6,7,8-tetrahydroimidazo[1,2-a]pyridin-2-yl]-benzimidazol-1-yl]-methyl]-1,r-biphenyl]-2-carboxylicacid,2-butyl-6-(l-methoxy-1-methylethyl)-2-[2′-)IH-tetrazol-5-yl)biphenyl-4-ylmethyl]guinazolin-4(3H)-one,3-[2′-carboxybiphenyl-4-yl)methyl]-2-cyclopropyl-7-methyl-3H-imidazo[4,5-b]pyridine,2-butyl-4-chloro-1-[(2′-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-carboxylicacid, 2-butyl-4-chloro-1-[[2′-(1H-tetrazol-5-yl) [1,1′-biphenyl]-4-yl]methyl]-1H-imidazole-5-carboxylicacid-1-(ethoxycarbonyl-oxy)ethyl ester potassium salt, dipotassium2-butyl-4-(methylthio)-1-[[2-[[[(propylamino)carbonyl]amino]-sulfonyl](1,1′-biphenyl)-4-yl]methyl]-1H-imidazole-5-carboxylate,methyl-2-[[4-butyl-2-methyl-6-oxo-5-[[2′-(1H-tetrazol-5-yl)-[1,1′-biphenyl]-4-yl]methyl]-1-(6H)-pyrimidinyl]methyl]-3-thiophencarboxylate,5-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-2-]2-(1H-tetrazol-5-ylphenyl)]pyridine, 6-butyl-2-(2-phenylethyl)-5 [[2′-(1H-tetrazol-5-yl)[1,1′-biphenyl]-4-methyl]pyrimidin-4-(3H)-one D, Llysine salt,5-methyl-7-n-propyl-8-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-[1,2,4]-triazolo[1,5-c]pyrimidin-2(3H)-one,2,7-diethyl-5-[[2′-(5-tetrazoly)biphenyl-4-yl]methyl]-5H-pyrazolo[1,5-b][1,2,4]triazolepotassium salt,2-[2-butyl-4,5-dihydro-4-oxo-3-[2′-(1H-tetrazol-5-yl)-4-biphenylmethyl]-3H-imidazol[4,5-c]pyridine-5-ylmethyl]benzoic acid, ethyl ester, potassium salt,3-methoxy-2,6-dimethyl-4-[[2′(1H-tetrazol-5-yl)-1,1′-biphenyl-4-yl]methoxy]pyridine,2-ethoxy-1-[[2′-(5-oxo-2,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylic acid, 1-[N-(2′-(1H-tetrazol-5-yl)biphenyl-4-yl-methyl)-N-valerolylaminomethyl)cyclopentane-1-carboxylicacid, 7-methyl-2n-propyl-3-[[2′1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3H-imidazo[4,5-6]pyridine,2-[5-[(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine-3-yl)methyl]-2-quinolinyl]sodiumbenzoate, 2-butyl-6-chloro-4-hydroxymethyl-5-methyl-3-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]pyridine,2-[[[2-butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol-5-yl]methyl]amino]benzoic acidtetrazol-5-yl)biphenyl-4-yl]methyl]pyrimidin-6-one,4(S)-[4-(carboxymethyl)phenoxy]-N-[2(R)-[4-(2-sulfobenzamido)imidazol-1-yl]octanoyl]-L-proline,1-(2,6-dimethylphenyl)-4-butyl-1,3-dihydro-3-[[6-[2-(1H-tetrazol-5-yl)phenyl]-3-pyridinyl]methyl]-2H-imidazol-2-one,5,8-ethano-5,8-dimethyl-2-n-propyl-5,6,7,8-tetrahydro-1-[[2′1Htetrazol-5-yl)biphenyl-4-yl]methyl]-1H,4H-1,3,4a,8a-tetrazacyclopentanaphthalene-9-one,4-[1-[2′-(1,2,3,4-tetrazol-5-yl)biphen-4-yl)methylamino]-5,6,7,8-tetrahydro-2-trifylquinazoline,2-(2-chlorobenzoyl)imino-5-ethyl-3-[2′-(1H-tetrazole-5-yl)biphenyl-4-yl)methyl-1,3,4-thiadiazoline,2-[5-ethyl-3-[2-(1H-tetrazole-5-yl)biphenyl-4-yl]methyl-1,3,4-thiazoline-2-ylidene]aminocarbonyl-1-cyclopentencarboxylicacid dipotassium salt, and2-butyl-4-[N-methyl-N-(3-methylcrotonoyl)amino]-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1 H-imidzole-5-carboxylic acid1-ethoxycarbonyloxyethyl ester, those disclosed in patent publicationsEP475206, EP497150, EP539086, EP539713, EP535463, EP535465, EP542059,EP497121, EP535420, EP407342, EP415886, EP424317, EP435827, EP433983,EP475898, EP490820, EP528762, EP324377, EP323841, EP420237, EP500297,EP426021, EP480204, EP429257, EP430709, EP434249, EP446062, EP505954,EP524217, EP514197, EP514198, EP514193, EP514192, EP450566, EP468372,EP485929, EP503162, EP533058, EP467207 EP399731 , EP399732, EP412848,EP453210, EP456442, EP470794, EP470795, EP495626, EP495627, EP499414,EP499416, EP499415, EP511791, EP516392, EP520723, EP520724, EP539066,EP438869, EP505893, EP530702, EP400835, EP400974, EP401030, EP407102,EP411766, EP409332, EP412594, EP419048, EP480659, EP481614, EP490587,EP467715, EP479479, EP502725, EP503838, EP505098, EP505111 EP513,979EP507594, EP510812, EP511767, EP512675, EP512676, EP512870, EP517357,EP537937, EP534706, EP527534, EP540356, EP461040, EP540039, EP465368,EP498723, EP498722, EP498721, EP515265, EP503785, EP501892, EP519831,EP532410, EP498361, EP432737, EP504888, EP508393, EP508445, EP403159,EP403158, EP425211, EP427463, EP437103, EP481448, EP488532, EP501269,EP500409, EP540400, EP005528, EP028834, EP028833, EP411507, EP425921,EP430300, EP434038, EP442473, EP443568, EP445811, EP459136, EP483683,EP518033, EP520423, EP531876, EP531874, EP392317, EP468470, EP470543,EP502314, EP529253, EP543263, EP540209, EP449699, EP465323, EP521768,EP415594, WO92/14468, WO93/08171, WO93/08169, WO91/00277, WO91/00281 ,WO91/14367, WO92/00067, WO92/00977, WO92/20342, WO93/04045, WO93/04046,WO91/15206, WO92/14714, WO92/09600, WO92/16552, WO93/05025, WO93/03018,WO91/07404, WO92/02508, WO92/13853, WO91/19697, WO91/11909, WO91/12001,WO91/11999, WO91/15209, WO91/15479, WO92/20687, WO92/20662, WO92/20661,WO93/01177, WO91/14679, WO91/13063, WO92/13564, WO91/17148, WO91/18888,WO91/19715, WO92/02257, WO92/04335, WO92/05161, WO92/07852, WO92/15577,WO93/03033, WO91/16313, WO92/00068, WO92/02510, WO92/09278, WO9210179,WO92/10180, WO92/10186, WO92/10181, WO92/10097, WO92/10183, WO92/10182,WO92/10187, WO92/10184, WO92/10188, WO92/10180, WO92/10185, WO92/20651,WO93/03722, WO93/06828, WO93/03040, W092/19211, WO92/22533, WO92/06081,WO92/05784, WO93/00341, WO92/04343, WO92/04059, U.S. Pat. No. 5,104,877,U.S. Pat. No. 5,187,168, U.S. Pat. No. 5,149,699, U.S. Pat. No.5,185,340, U.S. Pat. No. 4,880,804, U.S. Pat. No. 5,138,069, U.S. Pat.No. 4,916,129, U.S. Pat. No. 5,153,197, U.S. Pat. No. 5,173,494, U.S.Pat. No. 5,137,906, U.S. Pat. No. 5,155,126, U.S. Pat. No. 5,140,037,U.S. Pat. No. 5,137,902, U.S. Pat. No. 5,157,026, U.S. Pat. No.5,053,329, U.S. Pat. No. 5,132,216, U.S. Pat. No. 5,057,522, U.S. Pat.No. 5,066,586, U.S. Pat. No. 5,089,626, U.S. Pat. No. 5,049,565, U.S.Pat. No. 5,087,702, U.S. Pat. No. 5,124,335, U.S. Pat. No. 5,102,880,U.S. Pat. No. 5,128,327, U.S. Pat. No. 5,151,435, U.S. Pat. No.5,202,322, U.S. Pat. No. 5,187,159, U.S. Pat. No. 5,198,438, U.S. Pat.No. 5,182,288, U.S. Pat. No. 5,036,048, U.S. Pat. No. 5,140,036, U.S.Pat. No. 5,087,634, U.S. Pat. No. 5,196,537, U.S. Pat. No. 5,153,347,U.S. Pat. No. 5,191,086, U.S. Pat. No. 5,190,942, U.S. Pat. No.5,177,097, U.S. Pat. No. 5,212,177, U.S. Pat. No. 5,208,234, U.S. Pat.No. 5,208,235, U.S. Pat. No. 5,212,195, U.S. Pat. No. 5,130,439, U.S.Pat. No. 5,045,540, U.S. Pat. No. 5,041,152, and U.S. Pat. No. 5210204,and pharmaceutically acceptable salts and esters thereof; α/β adrenergicblockers such as nipradilol, arotinolol, amosulalol, bretylium tosylate(CAS RN: 61-75-6), dihydroergtamine mesylate (such as ergotaman-3′, 6′,18-trione,9,-10-dihydro-12′-hydroxy-2′-methyl-5′-(phenylmethyl)-,(5′(α))-,monomethanesulfonate, e.g., DHE 45® Injection, Novartis), carvedilol(such as(±)-1-(Carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol,e.g., Coreg®, SmithKline Beecham), labetalol (such as5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl) amino]ethyl]salicylamidemonohydrochloride, e.g., Normodyne®, Schering), bretylium tosylate(Benzenemethanaminium, 2-bromo-N-ethyl-N,N-dimethyl-, salt with4-methylbenzenesulfonic acid (1:1) CAS RN 61-75-6), phentolaminemesylate (Phenol,3-[[(4,5-dihydro-1H-imidazol-2-yl)methyl](4-methylphenyl)amino]-,monomethanesulfonate (salt) CAS RN 65-28-1), solypertine tartrate(5H-1,3-Dioxolo[4,5-f]indole,7-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-,(2R,3R)-2,3-dihydroxybutanedioate (1:1) CAS RN 5591-43-5), zolertinehydrochloride (Piperazine, 1-phenyl4-[2-(1H-tetrazol-5-yl)ethyl]-,monohydrochloride (8Cl, 9Cl) CAS RN 7241-94-3) and the like; aadrenergic receptor blockers, such as alfuzosin (CAS RN:81403-68- 1 ),terazosin, urapidil, prazosin (Minipress®), tamsulosin, bunazosin,trimazosin, doxazosin, naftopidil, indoramin, WHP 164, XENOIO,fenspiride hydrochloride (which may be prepared as disclosed in U.S.Pat. No. 3,399,192), proroxan (CAS RN 33743-96-3), and labetalolhydrochloride and combinations thereof; a 2 agonists such as methyldopa,methyldopa HCL, lofexidine, tiamenidine, moxonidine, rilmenidine,guanobenz, and the like; aldosterone inhibitors, and the like; renininhibitors including Aliskiren (SPPIOO; Novartis/Speedel);angiopoietin-2-binding agents such as those disclosed in WO03/030833;anti-angina agents such as ranolazine (hydrochloride 1-Piperazineacetamide,N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-,dihydrochloride CAS RN 95635-56-6), betaxolol hydrochloride (2-Propanol,1-[4-[2 (cyclopropylmethoxy)ethyl]phenoxy]-3-[(1-methylethyl)amino]-,hydrochloride CAS RN 63659-19-8), butoprozine hydrochloride (Methanone,[4-[3(dibutylamino)propoxy]phenyl](2-ethyl-3-indolizinyl)-,monohydrochloride CAS RN 62134-34-3), cinepazetmaleatel-Piperazineacetic acid, 4-[1-oxo-3-(3,4,5- trimethoxyphenyl)-2-propenyl]-, ethyl ester, (2Z)-2-butenedioate (1:1) CAS RN 50679-07-7),tosifen (Benzenesulfonamide,4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]arnino]carbonyl]-CAS RN32295-184), verapamilhydrochloride (Benzeneacetonitrile,α-[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-α-(1-methylethyl)-,monohydrochloride CAS RN 152-114), molsidomine (1,2,3-Oxadiazolium,5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt CAS RN25717-80-0), and ranolazine hydrochloride (1-Piperazineacetamide,N-(2,6-dimethylphenyl)4-[2-hydroxy-3-(2-meth-oxyphenoxy)propyl]-,dihydrochloride CAS RN 95635-56-6); tosifen (Benzenesulfonamide,4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]- CAS RN32295-184); adrenergic stimulants such as guanfacine hydrochloride (suchas N-amidino-2-(2,6-dichlorophenyl) acetamide hydrochloride, e.g.,Tenex® Tablets available from Robins); methyldopa-hydrochlorothiazide(such as levo-3-(3,4-dihydroxyphenyl)-2-methylalanine) combined withHydrochlorothiazide (such as 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide, e.g., the combinationas, e.g., Aldoril® Tablets available from Merck),methyldopa-chlorothiazide (such as6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide andmethyldopa as described above, e.g., Aldoclor®, Merck), clonidinehydrochloride (such as 2-(2,6-dichlorophenylamino)-2-imidazolinehydrochloride and chlorthalidone (such as2-chloro-5-(1-hydroxy-3-oxo-1-isoindolinyl) benzenesulfonamide), e.g.,Combipres®, Boehringer Ingelheim), clonidine hydrochloride (such as2-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride, e.g.,Catapres®, Boehringer Ingelheim), clonidine (1H-Imidazol-2-amine,N-(2,6- dichlorophenyl)4,5-dihydro-CAS RN 4205-90-7), Hyzaar (Merck; acombination of losartan and hydrochlorothiazide), Co-Diovan (Novartis; acombination of valsartan and hydrochlorothiazide, Lotrel (Novartis; acombination of benazepril and amlodipine) and Caduet (Pfizer; acombination of amlodipine and atorvastatin), and those agents disclosedin U.S. PAT. NO. 20030069221.

1.2.2.11 Agents for the Treatment of Respiratory Disorders

The GCC agonist peptides described herein can be used in combinationtherapy with one or more of the following agents useful in the treatmentof respiratory and other disorders including but not limited to: (1)β-agonists including but not limited to: albuterol (PRO VENTIL®, S ALBUTAMOl®, VENTOLIN®), bambuterol, bitoterol, clenbuterol, fenoterol,formoterol, isoetharine (BRON OSOL®, BRON OMETER®), metaproterenol(ALUPENT®, METAPREL®), pirbuterol (MAXAIR®), reproterol, rimiterol,salmeterol, terbutaline (BRETHAIRE®, BRETHINE®, BRICANYL®), adrenalin,isoproterenol (ISUPREL®), epinephrine bitartrate (PRIMATENE®),ephedrine, orciprenline, fenoterol and isoetharine; (2) steroids,including but not limited to beclomethasone, beclomethasonedipropionate, betamethasone, budesonide, bunedoside, butixocort,dexamethasone, flunisolide, fluocortin, fluticasone, hydrocortisone,methyl prednisone, mometasone, predonisolone, predonisone, tipredane,tixocortal, triamcinolone, and triamcinolone acetonide; (3)2-agonist-corticosteroid combinations [e.g., salmeterol-fluticasone(ADVAIR®), formoterol-budesonid (SYMBICORT®]; (4) leukotriene D4receptor antagonists/leukotriene antagonists/LTD4 antagonists (i.e., anycompound that is capable of blocking, inhibiting, reducing or otherwiseinterrupting the interaction between leukotrienes and the Cys LTIreceptor) including but not limited to: zafhiukast, montelukast,montelukast sodium (SINGULAIR®), pranlukast, iralukast, pobilukast,SKB-106,203 and compounds described as having LTD4 antagonizing activitydescribed in U.S. Pat. No. 5,565,473; (5) 5-lipoxygenase inhibitorsand/or leukotriene biosynthesis inhibitors [e.g., zileuton and BAY 1005(CA registry 128253-31 -6)]; (6) histamine HI receptorantagonists/antihistamines (i.e., any compound that is capable ofblocking, inhibiting, reducing or otherwise interrupting the interactionbetween histamine and its receptor) including but not limited to:astemizole, acrivastine, antazoline, azatadine, azelastine, astamizole,bromopheniramine, bromopheniramine maleate, carbinoxamine, carebastine,cetirizine, chlorpheniramine, chloropheniramine maleate, cimetidineclemastine, cyclizine, cyproheptadine, descarboethoxyloratadine,dexchlorpheniramine, dimethindene, diphenhydramine, diphenylpyraline,doxylamine succinate, doxylarnine, ebastine, efletirizine, epinastine,famotidine, fexofenadine, hydroxyzine, hydroxyzine, ketotifen,levocabastine, levocetirizine, levocetirizine, loratadine, meclizine,mepyramine, mequitazine, methdilazine, mianserin, mizolastine,noberastine, norasternizole, noraztemizole, phenindamine, pheniramine,picumast, promethazine, pynlamine, pyrilamine, ranitidine, temelastine,terfenadine, trimeprazine, tripelenamine, and triprolidine; (7) ananticholinergic including but not limited to: atropine, benztropine,biperiden, flutropium, hyoscyamine (e.g. Levsin®; Levbid®; Levsin/SL®,Anaspaz®, Levsinex timecaps®, NuLev®), ilutropium, ipratropium,ipratropium bromide, methscopolamine, oxybutinin, rispenzepine,scopolamine, and tiotropium; (8) an anti-tussive including but notlimited to: dextromethorphan, codeine, and hydromorphone; (9) adecongestant including but not limited to: pseudoephedrine andphenylpropanolamine; (10) an expectorant including but not limited to:guafenesin, guaicolsulfate, terpin, ammonium chloride, glycerolguaicolate, and iodinated glycerol; (11) a bronchodilator including butnot limited to: theophylline and aminophylline; (12) ananti-inflammatory including but not limited to: fluribiprofen,diclophenac, indomethacin, ketoprofen, S-ketroprophen, tenoxicam; (13) aPDE (phosphodiesterase) inhibitor including but not limited to thosedisclosed herein; (14) a recombinant humanized monoclonal antibody [e.g.xolair (also called omalizumab), rhuMab, and talizumab]; (15) ahumanized lung surfactant including recombinant forms of surfactantproteins SP-B, SP-C or SP-D [e.g. SURFAXIN®, formerly known as dsc-104(Discovery Laboratories)], (16) agents that inhibit epithelial sodiumchannels (ENaC) such as amiloride and related compounds; (17)antimicrobial agents used to treat pulmonary infections such asacyclovir, amikacin, amoxicillin, doxycycline, trimethoprinsulfamethoxazole, amphotericin B, azithromycin, clarithromycin,roxithromycin, clarithromycin, cephalosporins (ceffoxitin, cefmetazoleetc), ciprofloxacin, ethambutol, gentimycin, ganciclovir, imipenem,isoniazid, itraconazole, penicillin, ribavirin, rifampin,rifabutin,amantadine, rimantidine, streptomycin, tobramycin, andvancomycin; (18) agents that activate chloride secretion through Ca++dependent chloride channels (such as purinergic receptor (P2Y(2)agonists); (19) agents that decrease sputum viscosity, such as humanrecombinant DNase 1, (Pulmozyme®); (20) nonsteroidal anti-inflammatoryagents (acemetacin, acetaminophen, acetyl salicylic acid, alclofenac,alminoprofen, apazone, aspirin, benoxaprofen, bezpiperylon, bucloxicacid, carprofen, ciidanac, diclofenac, diclofenac, diflunisal,diflusinal, etodolac, fenbufen, fenbufen, fenclofenac, fenclozic acid,fenoprofen, fentiazac, feprazone, flufenamic acid, flufenisal,flufenisal, fluprofen, flurbiprofen, flurbiprofen, furofenac, ibufenac,ibuprofen, indomethacin, indomethacin, indoprofen, isoxepac, isoxicam,ketoprofen, ketoprofen, ketorolac, meclofenamic acid, meclofenamic acid,mefenamic acid, mefenamic acid, miroprofen, mofebutazone, nabumetoneoxaprozin, naproxen, naproxen, niflumic acid , oxaprozin, oxpinac,oxyphenbutazone, phenacetin, phenylbutazone, phenylbutazone, piroxicam,piroxicam, pirprofen, pranoprofen, sudoxicam, tenoxican, sulfasalazine,sulindac, sulindac, suprofen, tiaprofenic acid, tiopinac, tioxaprofen,tolfenamic acid, tolmetin, tolmetin, zidometacin, zomepirac, andzomepirac); and (21) aerosolized antioxidant therapeutics such asS-Nitrosoglutathione.

1.2.2.12 Anti-Diabetic Agents

The GCC agonist peptides described herein can be used in therapeuticcombination with one or more anti-diabetic agents, including but notlimited to: PPARy agonists such as glitazones (e.g., WAY-120,744, AD5075, balaglitazone, ciglitazone, darglitazone (CP-86325, Pfizer),englitazone (CP-68722, Pfizer), isaglitazone (MIT/J&J), MCC-555(Mitsibishi disclosed in U.S. PAT. NO. 5594016), pioglitazone (such assuch as Actos™ pioglitazone; Takeda), rosiglitazone (Avandia™;SmithKline Beecham), rosiglitazone maleate, troglitazone (Rezulin®, disclosedin us4,572,912), rivoglitazone (CS-Ol 1, Sankyo), GL-262570 (GlaxoWelcome), BRL49653 (disclosed in WO98/05331), CLX-0921, 5-BTZD, GW-0207,LG-100641, JJT-501 (JPNT/P&U), L-895645 (Merck), R- l 19702(Sankyo/Pfizer), NN-2344 (Dr. Reddy/NN), YM-440 (Yamanouchi), LY-300512,LY-519818, R483 (Roche), T131 (Tularik), and the like and compoundsdisclosed in U.S. Pat. No. 4,687,777, U.S. Pat. No. 5,002,953, U.S. Pat.No. 5,741,803, U.S. Pat. No. 5,965,584, U.S. Pat. No. 6,150,383, U.S.Pat. No. 6,150,384, U.S. Pat. No. 6,166,042, U.S. Pat. No. 6,166,043,U.S. Pat. No. 6,172,090, U.S. Pat. No. 6,211,205, U.S. Pat. No.6,271,243, U.S. Pat. No. 6,288,095, U.S. Pat. No. 6,303,640, U.S. Pat.No. 6,329,404, U.S. Pat. No. 5,994,554, WO97/10813, WO97/27857,WO97/28115, WO97/28137, WO97/27847, WO00/76488, WO03/000685,WO03/027112, WO03/035602, WO03/048130, WO03/055867, and pharmaceuticallyacceptable salts thereof; biguanides such as metformin hydrochloride(N,N-dimethylimidodicarbonimidic diamide hydrochloride, such asGlucophage™, Bristol-Myers Squibb); metformin hydrochloride withglyburide, such as Glucovance™, Bristol-Myers Squibb); buformin(Imidodicarbonimidic diamide, N-butyl-); etoformine(1-Butyl-2-ethylbiguanide, Schering A. G.); other metformin salt forms(including where the salt is chosen from the group of, acetate,benzoate, citrate, ftimarate, embonate, chlorophenoxyacetate, glycolate,palmoate, aspartate, methanesulphonate, maleate,parachlorophenoxyisobutyrate, formate, lactate, succinate, sulphate,tartrate, cyclohexanecarboxylate, hexanoate, octanoate, decanoate,hexadecanoate, octodecanoate, benzenesulphonate, trimethoxybenzoate,paratoluenesulphonate, adamantanecarboxylate, glycoxylate, glutarnate,pyrrol idonecarboxylate, naphthalenesulphonate, 1-glucosephosphate,nitrate, sulphite, dithionate and phosphate), and phenformin; proteintyrosine phosphatase-IB (PTP-IB) inhibitors, such as A-401,674, KR61639, OC-060062, OC-83839, OC-297962, MC52445, MC52453, ISIS 113715,and those disclosed in WO99/585521, WO99/58518, WO99/58522, WO99/61435,WO03/032916, WO03/032982, WO03/041729, WO03/055883, WO02/26707,WO02/26743, JP2002114768, and pharmaceutically acceptable salts andesters thereof; sulfonylareas such as acetohexamide (e.g. Dymelor, EliLilly), carbutamide, chlorpropamide (e.g. Diabinese®, Pfizer),gliamilide (Pfizer), gliclazide (e.g. Diamcron, Servier Canada Inc),glimepiride (e.g. disclosed in U.S. Pat. No. 4,379,785, such as Amaryl,Aventis), glipentide, glipizide (e.g. Glucotrol or Glucotrol XL ExtendedRelease, Pfizer), gliquidone, glisolamide, glyburide/glibenclamide (e.g.Micronase or Glynase Prestab, Pharmacia & Upjohn and Diabeta, Aventis),tolazamide (e.g. Tolinase), and tolbutamide (e.g. Orinase), andpharmaceutically acceptable salts and esters thereof; meglitinides suchas repaglinide (e.g. Pranidin®, Novo Nordisk), KAD1229 (PF/Kissei), andnateglinide (e.g. Starlix®, Novartis), and pharmaceutically acceptablesalts and esters thereof; a glucoside hydrolase inhibitors (or glucosideinhibitors) such as acarbose (e.g. Precose™, Bayer disclosed in U.S.Pat. No. 4,904,769), miglitol (such as GLYSET™, Pharmacia & Upjohndisclosed in U.S. Pat. No. 4,639,436), camiglibose (Methyl6-deoxy-6-[(2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidino]-alpha-D-glucopyranoside,Marion Merrell Dow), voglibose (Takeda), adiposine, emiglitate,pradimicin-Q, salbostatin, CKD-71 1 , MDL-25,637, MDL-73,945, and MOR14, and the compounds disclosed in U.S. Pat. No. U.S. Pat. No.4,062,950, U.S. Pat. No. 4,174,439, U.S. Pat. No. 4,254,256, U.S. Pat.No. 4,701,559, U.S. Pat. No. 4,639,436, U.S. Pat. No. 5,192,772, U.S.Pat. No. 4,634,765, U.S. Pat. No. 5,157,116, U.S. Pat. No. 5,504,078,U.S. Pat. No. 5,091,418, U.S. Pat. No. 5,217,877, U.S. Pat. No. 51,091and WOO 1/47528 (polyamines); α-amylase inhibitors such as tendamistat,trestatin, and A1-3688, and the compounds disclosed in U.S. Pat. No.4,451,455, U.S. Pat. No. 4,623,714, and U.S. Pat. No. 4,273,765; SGLT2inhibtors including those disclosed in U.S. Pat. No. 6,414,126 and U.S.Pat. No. 6,515,117; an aP2 inhibitor such as disclosed in U.S. Pat. No.6,548,529; insulin secreatagogues such as linogliride, A-4166, forskilin, dibutyrl cAMP, isobutylmethylxanthine (IBMX), and pharmaceuticallyacceptable salts and esters thereof; fatty acid oxidation inhibitors,such as clomoxir, and etomoxir, and pharmaceutically acceptable saltsand esters thereof; A2 antagonists, such as midagl izole, isaglidole,deriglidole, idazoxan, earoxan, and fluparoxan, and pharmaceuticallyacceptable salts and esters thereof; insulin and related compounds (e.g.insulin mimetics) such as biota, LP-100, novarapid, insulin detemir,insulin lispro, insulin glargine, insulin zinc suspension (lente andultralente), Lys-Pro insulin, GLP-I (1-36) amide, GLP-I (73-7)(insulintropin, disclosed in U.S. Pat. No. 5,614,492), LY-315902(Lilly), GLP-I (7-36)-NH2), AL-401 (Autoimmune), certain compositions asdisclosed in U.S. Pat. No. 4,579,730, U.S. Pat. No. 4,849,405, U.S. Pat.No. 4,963,526, U.S. Pat. No. 5,642,868, U.S. Pat. No. 5,763,396, U.S.Pat. No. 5,824,638, U.S. Pat. No. 5,843,866, U.S. Pat. No. 6,153,632,U.S. Pat. No. 6,191,105, and WO 85/05029, and primate, rodent, or rabbitinsulin including biologically active variants thereof including allelicvariants, more preferably human insulin available in recombinant form(sources of human insulin include pharmaceutically acceptable andsterile formulations such as those available from Eli Lilly(Indianapolis, Ind. 46285) as Humulin™ (human insulin rDNA origin), alsosee the THE PHYSICIAN'S DESK REFERENCE, 55.sup.th Ed. (2001) MedicalEconomics, Thomson Healthcare (disclosing other suitable humaninsulins); non-thiazolidinediones such as JT-501 and farglitazar(GW-2570/GI-262579), and pharmaceutically acceptable salts and estersthereof; PPARα/γ dual agonists such as AR-H039242 (Aztrazeneca),GW-409544 (Glaxo-Wellcome), BVT-142, CLX-0940, GW-1536, GW-1929,GW-2433, KRP-297 (Kyorin Merck; 5-[(2,4-Dioxothiazolidinyl)methyl]methoxy-N-[[4-(trifluoromethyl)phenyl]methyljbenzamide),L-796449. LR-90, MK-0767 (Merck/Kyorin/Banyu), SB 219994, muraglitazar(BMS), tesaglitzar (Astrazeneca), reglitazar (JTT-501) and thosedisclosed in WO99/16758, WO99/ 19313, WO99/20614, WO99/38850,WO00/23415, WO00/23417, WO00/23445, WO00/50414, WOO 1/00579, WOO1/79150, WO02/062799, WO03/004458, WO03/016265, WO03/01 8010,WO03/033481 , WO03/033450, WO03/033453, WO03/043985, WO 031053976, U.S.application Ser. No. 09/664,598, filed Sep. 18, 2000, Murakami et al.Diabetes 47,1841 - 1 847 (1998), and pharmaceutically acceptable saltsand esters thereof; other insulin sensitizing drugs; VPAC2 receptoragonists; GLK modulators, such as those disclosed in WO03/01 5774;retinoid modulators such as those disclosed in WO03/000249; GS 3/GS 3inhibitors such as4-[2-(2-bromophenyl)-4-(4-fluorophenyl-1H-imidazol-5-yl]pyridine andthose compounds disclosed in WO03/024447, WO03/037869, WO03/037877,WO03/037891 , WO03/068773, EP 1295884, EP 1295885, and the like;glycogen phosphorylase (HGLPa) inhibitors such as CP-368,296. CP-316,819, BAYR3401, and compounds disclosed in WOO 1 /94300, WO02/20530,WO03/037864, and pharmaceutically acceptable salts or esters thereof;ATP consumption promoters such as those disclosed in WO03/007990; TRB3inhibitors; vanilloid receptor ligands such as those disclosed inWO03/049702; hypoglycemic agents such as those disclosed in WO03/015781and WO03/040114; glycogen synthase kinase 3 inhibitors such as thosedisclosed in WO03/035663 agents such as those disclosed in WO99/51225,US20030134890, WO01/24786, and WO03/059870; insulin-responsive DNAbinding protein-1 (IRDBP-1) as disclosed in WO03/057827, and the like;adenosine A2 antagonists such as those disclosed in WO03/035639,WO03/035640, and the like; PPAR5 agonists such as GW 501516, GW 590735,and compounds disclosed in JP 10237049 and WO02/14291; dipeptidylpeptidase IV (DP-IV) inhibitors, such as isoleucine thiazolididc,NVP-DPP728A(1-[[[2-[(5-cyanopyridin-2-yl)amino]ethyl]amino]acetyl]-2-cyano-(S)-pyrrolidine,disclosed by Hughes et al, Biochemistry, 38(36), 1 1597- 1 1603, 1999),P32/98, NVP-LAF-237, P3298, TSL225 (tryptophyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid, disclosed by Yamada et al,Bioorg. & Med. Chem. Lett. 8 ( 1 998) 1537- 1540), valine pyrrolidide,TMC-2A/2B/2C, CD-26 inhibitors, FE999011, P93 10/ 364, VIP 0177, DPP4,SDZ 274-444, 2-cyanopyrroIidides and 4-cyanopyrrolidides as disclosed byAshworth et al, Bioorg. & Med. Chem. Lett., Vol. 6, No. 22, pp 1163-1166and 2745-2748 ( 1996) ,and the compounds disclosed in U.S. Pat. No.6,395,767, U.S. Pat. No. 6,573,287, U.S. Pat. No. 6,395,767 (compoundsdisclosed include BMS-477118, BMS-471211 and BMS 538,305), WO99/38501,WO99/46272, WO99/67279, WO99/67278, WO99/61431 WO03/004498, WO03/004496,EP 1258476, WO02/083128, WO02/062764, WO03/000250, WO03/002530,WO03/002531, WO03/002553, WO03/002593, WO03/000180, and WO03/000181 ;GLP-I agonists such as exendin-3 and exendin-4 (including the 39 aapolypeptide synthetic exendin-4 called Exenatide®), and compoundsdisclosed in US2003087821 and NZ 504256, and pharmaceutically acceptablesalts and esters thereof; peptides including amlintide andSymlin®(pramlintide acetate); and glycokinase activators such as thosedisclosed in US2002103199 (fused heteroaromatic compounds) andWO02/48106 (isoindolin-1-one-substituted propionamide compounds).

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties. However, where apatent, patent application, or publication containing expressdefinitions is incorporated by reference, those express definitionsshould be understood to apply to the incorporated patent, patentapplication, or publication in which they are found, and not to theremainder of the text of this application. In the case of conflict, thepresent specification, including definitions, will control. Thereferences cited herein are not admitted to be prior art to theinvention.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description is intended to illustrate and not limit the scopeof the invention. It will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted withoutdeparting from the scope of the invention, and further that otheraspects, advantages and modifications will be apparent to those skilledin the art to which the invention pertains.

EXAMPLES Example 1 Preparation of Side-Chain Protected Fragments ofSP-304 Attachment Fmoc-AA-OH to 2-ClTrt Resin

2-ClTrt resin (10 g, substitution =1.0 mmol/g resin) was suspended in100 mL of dichloromethane (DCM) for 5 minutes, and then drained. Theesterification was performed using 1.5 equiv. of Fmoc-amino acid and 1.7equiv. Diisopropylethylamine (DIEA) in 80 mL of DCM (with minimumquantity of dimethylformamide (DMF) to dissolve the amino acidcompletely) for 2 hours. The resulting resin was washed with 60 mL ofDCM and endcapped with 60 mL of DIEA/methanol (1:9, v/v) solution for 30minutes. The loaded resin was then washed with DCM (6 vol.) for 2 times,DMF (6 vol.) for 3 times and methyl t-butylether (MTBE) (6 vol.) for 3times, and dried under high vacuum. The substitution of theFmoc-protected resin was determined by Fmoc release assay. Finally, theFmoc group was deprotected with a mixture of 5% piperidine, 1%1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) and 1% N-Hydroxybenzotriazole(HOBt) in DMF (10 vol.) for 2 times and the resin was washed and driedunder high vacuum to give the final resin for peptide synthesis. Theresults of the experiments are listed in Table VIII below.

TABLE VIII Preparation of H-Gly-2ClTrt and H-Leu-2ClTrt resin SynthesisSubstitution of the Yield Amino acid 2-ClTrt scale loaded resin from(Weight, resin (mmol) Fmoc release assay % yield) H-Gly-2ClTrt resin4.16 0.57 mmol/g resin    6 g, 82% H-Leu-2ClTrt resin 5.20 0.81 mmol/gresin   6.4 g, 100% H-Gly-2ClTrt resin 300 0.80 mmol/g resin 328.6 g,88% H-Leu-2ClTrt resin 300 0.75 mmol/g resin 338.5 g, 84%

Synthesis of Side-Chain-Protected Fragments A and B

H-Gly-2-ClTrt resin or H-Leu-2-ClTrt resin was suspended in DMF (10vol.) for 20 minutes, then drained. The resulting resin was washed withDMF ( 10 vol.) for 5 minutes. The chain assembly was conducted using thestandard Fmoc chemistry. Generally, 1.5 equiv. of Fmoc amino acid and1.5 equiv. of HOBt were dissolved in DMF (4.5 vol.), followed byaddition of 1.5 equiv. of DIEA. Then, the resulting solution was cooledto below 5° C. with an ice water bath, and activated by addition of 1.5equiv. of HBTU. DCM (1.5 vol.) was added to the resin, followed byaddition of the activated Fmoc amino acid solution. The resultingmixture was stirred at room temperature for 2 hours and the completionof the acylation was monitored by Kaiser Test. If Kaiser Test indicatedthe presence of unreacted amine after 2 hours, recoupling with the sameprotocol using 1 .0 equiv. of Fmoc amino acid, 1.0 equiv. of HOBt and1.0 equiv. of DIEA was required. Capping was generally achieved byacetylating the unreacted amine with a mixture of aceticanhydride/pyridine/DMF solution. The peptide sequence was assembled byrepeating the above capping procedure with the corresponding Fmoc-aminoacid derivatives in the sequence from C- to N-terminal. The coupling ofFmoc-Cys(Trt)-OH or Fmoc-Cys(Acm)-OH residue was achieved by using 2.0equiv. of Fmoc-Cys(Trt)-OH or Fmoc-Cys(Acm)-OH, 2.0 of equiv. HOBt and2.0 equiv. of DIC in situ activation in DCM/DMF protocol to minimizeracemization of cysteine.

After completion of the synthetic step, the peptide resin was thoroughlywashed with DMF (10 vol.), MTBE (1 0 vol.), DMF (10 vol. 3 times) andMTBE (10 vol. 3 times) and subsequently dried in a vacuum oven to aconstant weight.

The side-chain-protected peptide was cleaved from the resin using 1%TFA/DCM (10 vol.) for 3 times, 5 minutes for each time and the cleavagefractions were collected onto pyridine each time (1:1 volume ratio toTFA in each cleavage fraction). The peptide resin was washed with DCM(7.5 vol.). The fractions were combined and concentrated under vacuum to10% of the original volume, and the resulting solution was reconstitutedwith ethanol (3 vol.) and concentrated to 50% of the original volume.Finally, the peptide was precipitated out by addition of water (1 vol.).The solid was collected by vacuum filtration or centrifugation andwashed with water twice. The product was dried in vacuum to a constantweight and subjected to HPLC and ES-S analysis. The results of theexperiments are presented in Table IX below.

TABLE IX Preparation of Fragments A and B Synthesis Final peptide resinweight Yield of Fragment scale and yield from the weight obtained PurityFragment Masses Fragment (mmol) gain of the resin (% yield) (HPLC)Calculated/Found* FmocAA7-14OH (1) 3.4  9.5 g, 66.4% 3.337 g (61.3%)87.8% 1599.95/1598.86 BocAA1-6OH (2) 5.2 11.8 g, 76.3% 5.896 g (76.9%)94.6% 1474.80/1473.94 FmocAA7-14OH (1′) 200 479.7 g, 74.4%  213.7 g(66.8%) 90.1% 1599.95/1598.81 BocAA1-6OH (2′) 200 544.0 g, 101.3% 247.0g (83.7%) 94.2% 1474.80/1473.94 *Calculated = average molecular mass;Found = mono-isotopic mass by ES-MS

Example 2 Condensation of Fragments of SP-304 in Solution Synthesis ofFragment C: H-AA 15- 16QtBu (1-1)

A solution of Fmoc-Cys(Acm)-OH (124.38 gm, 0.3 mol), H-Leu-OtBu.HCl(67.12 gm, 0.3 mol), and HOBt (40.54 gm, 0.3 mol) in DMF (600 mL) wascooled to -5° C. 2-[1 H-Benzotriazole-1-yl]-1, 1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) (113.79 gm, 0.3 mol) was added and dissolvedcompletely. D1EA ( 183.1 mL, 1.05 mol) was added dropwise over a periodof 105 minutes at the same temperature with good stirring, keeping thepH of the mixture between 6 and 7. Stirring was continued for 15 minutesat 0° C. and the reaction was monitored with TLC. The reaction mixturewas diluted with ethyl acetate (EtOAc) (600 mL) and 5% H3PO4 (300 mL).The organic layer was separated and the aqueous layer was extracted withEtOAc (600 mL). The combined extracts were washed with 5% H₃PO₄ (2times), H₂O(1 time), saturated NaHCO₃ (3 times), H₂O (2 times), andbrine (2 times). The solution was dried over MgSO₄ anhydrous, filtered,and evaporated in vacuo to dryness. The product was recrystallized frompetroleum ether/EtOAc (3 : 1 ) and dried: 166.75 gm (yield 95.0%, purity99.0%).

Fmoc-Cys(Acm)-Leu-OtBu (166.75 gm, 0.277 mol) was dissolved in a 10%piperidine/DCM solution (810 mL) with stirring. The reaction wasmonitored with TLC. After the reaction was completed in 3 hours, thesolvent and the volatile materials were removed using a rotavap. Theoily material obtained was triturated with petroleum ether to remove theby-products by decantation. The residue, a syrup, was taken up in EtOAc,and washed with a mixture solution of aH₂PO₄/Na₂HPO₄(pH=6), thensaturated NaHCO₃, purified H₂O, and brine. The organic layer was driedover MgSO₄ anhydrous. Evaporation of the solvent and the volatilematerials yielded an oily product H-Cys(Acm)-Leu-OtBu (1-1) (73.23 gm,yield: 73.1%, purity: 98.0%).

Synthesis of Fragment B-C: HAA7-16QtBu (2-3)

A solution of Fmoc-AA7-140H (1′) ( 198.3 gm, 124.0 mmol), H-AA 15-16OtBu(1-1) (52.3 gm, 148.8 mmol) and Cl-HOBt (21.0 gm, 124.0 mmol) in DMF(2500 mL) was cooled to −5° C. HBTU (51.7 gm, 136.4 mmol) was added anddissolved completely. DIEA (54.1 mL, 310 mmol) was then added withstirring, keeping the pH of the mixture between 6 and 7. Stirring wascontinued for 30 minutes at 0-5° C. The reaction mixture was allowed towarm up to 20-27° C. slowly and stirring was continued for one and ahalf hours. Then, the mixture was poured into precooled ( 10-20 ° C.)0.5 N HCl aq. (20 L).The suspension was stored at 20-25 ° C. for 45minutes. The solid was collected by filtering the suspension through afritted-glass funnel of medium porosity and subsequent washing with 0.5N HCl aq.(3500 mL, 2 times), purified water (3500 mL), saturated NaHCO₃aq. (3500 mL, 2 times) and purified water (3500 mL, 2 times) and diethylether (2000 mL, 2 times). Finally, the wet, crude peptide FmocAA7-16OtBu(2-3) was dried in a desiccator under high vacuum at room temperature toyield product 238.22 gm (purity, 85.27%, yield 98.9%).

Synthesis of the Fully Protected A-B-C: BocAA1-16OtBu (3-3)

A solution of HAA7-16OtBu (2-3) (1 83.67 gm, 106.71 mmol), BocAA 1-6-OH(2′) (157.65 gm, 106.71 mmol), and 6-Cl-HOBt (18.141 gm, 106.71 mmol) inDMF (3 L) were cooled to at −3 to 0° C. HBTU (44.523 gm, 117.38 mmol)was added and dissolved completely. DIEA (55.8 mL, 320.13 mmol) was thenadded with stirring, keeping the pH of the mixture 6. Stirring wascontinued for 20 minutes at −5 to 0° C. The reaction mixture was allowedto warm up to 25° C. slowly and stirring was continued for 2.5 hours,followed by further addition of HBTU (4.048 gm, 10.671 mmol) and DIEA (2mL). Stirring was continued for another 1.5 hours. The resulting mixturewas poured into MeOH (15 L), and the precipitate was collected andwashed with MeOH/DMF mixture (5:1 , v/v) (2 times, 3 L), 0.1 N HCl (3 L,2 times), saturated NaHCO₃ (2 times), purified water (3 times), diethylether (2 times) and dried in vacuo to yield the product BocAA1-16OtBu(3-3) (278.0 gm, yield 82.0%. Note: the purity was determined after thedeprotection).

Synthesis of the Partially Protected Linear SP-304: HAA1-16OH (4-4)

A mixture of TFA/TIS/EDT (8:1:1, 2400 mL) was cooled to (0-5° C.) undernitrogen and Boc-AA1-16OtBu (3-3) (201 gm) was added in portions. Theresulting suspension was stirred at 0- 10° C. for 30 min, then thereaction solution was allowed to warm up to 20-25° C. with a water bath(10 minutes) and stirring was continued for additional 1 hr and 50 minat the same temperature. The reaction mixture was poured into pre-cooled(10° C.) MTBE (18 L). Some heat was evolved during the addition of thepeptide/TFA solution and the internal temperature went up to 25° C. Theresulting suspension was then stored in an ice-water bath (5-10° C.) for40 min. The precipitate was collected by filtration and washed with MTBE(2000 mL, 4 times), and dried in vacuum over P₂O₅, yielding 148.37 gm ofoff-white product, HAA 1-16OH (4-4) (purity: 62.23%, ES-MS, MW:calculated=1828.07, found=1826.67).

Example 3 Oxidative Cyclization and Purification of SP-304 byPolystyrenic Absorbent Resin

HAA1-16OH (4-4) (0.58 gm) was dissolved in 5 mL of acetonitrile anddiluted with 575 mL of purified water. The solution was adjusted to pH8-9 with 25% ammonia solution, and 3% hydrogen peroxide (0.58 mL) wasadded, then the reaction mixture was kept for an hour with monitoringthe disulfide formation by HPLC. Nitrogen was then passed through thereaction mixture and the solution was acidified to pH 3-4 with aceticacid (71.8% HPLC, recovery 98.5% estimated from peak area). Theresulting mixture was added 1 % iodine/ACN dropwise over a period of 10minutes with good stirring until the yellow color of the iodinepersisted. Stirring was continued for 30 minutes at 17-20° C. The iodinewas quenched by addition of 0.5 M ascorbic acid aq. until the yellowdisappeared. Then, the pH of the mixture was adjusted to 6-7 with 25%ammonia solution (51.0% HPLC, recovery 50% estimated from peak area).

The polystyrenic absorbent resin (D101 ) was packed to a 3(ID)×9(L) CMcolumn and well equilibrated with 6 column volume (CV) of ethanol, 4 CVof purified water, 2 CV of 5% HCl aq., 4 CV of purified water, 2 CV of2% NaOH aq. and 4 CV of purified water at the flow rate of 3 CV/hour.The oxidized peptide solution was then loaded to the column at 2 CV/h.After loading, the column was washed with 2 CV purified water at 2 CV/h.The elution was conducted by applying 80% ethanol aq. to the column at 2CV/h. The fractions with UV absorbents at 215 nm, was collected andcombined (125 mL). The combined fractions were then evaporated in vacuumto 10% of the original volume and the suspension was precipitated with125 mL of cold MTBE ( 10 vol). The solid was collected by filtration anddried in vacuum to yield the crude SP-304 (0.282 g, 55.0% HPLC).

Example 4 Oxidative Cyclization and Purification of SP-304 by RP-HPLC

Crude peptide (4-4), prepared as described in Example 2 was dissolved in10% ACN aqueous to an approximate concentration of 1.25 g/L withcontinuous stirring via a mechanical stirrer. The pH of the peptidesolution was adjusted to 8.5-9.0 with 20% ammonia aqueous and theresulting solution was stirred vigorously open to the atmosphere.Hydrogen peroxide (3%, 0.25 equiv.) was added and stirring was continuedat room temperature for 60 minutes. The HPLC analysis showed completeconsumption of the linear peptide. Then, the solution was acidified topH 3-4 with 10% AcOH aqueous. The resulting solution was diluted to aconcentration of about 1 g/L with purified water. Iodine (1.3% in ACN)was added in with vigorous stirring over a period of 10 minutes untilthe yellow color of the iodine persisted. At about half-hourly intervalssamples were taken from the mixture and analyzed by RP-HPLC. Themonocyclized peptide peak decreased gradually and a new peak (dicyclizedpeptide) emerged. Oxidation was complete when no monocyclized peptidepeak left. The excess iodine was neutralized by a small amount ofascorbic acid. The resulting solution was loaded on a C₁₈ RP-HPLC columnpacked with Kromasil 100 Å, 10 μm silica gel. After the dicyclizedpeptide solution was loaded, a 3 column volume of a solution of 90%mobile phase A (1.0%TEA, 0.5%H₃PO₄ in H₂O, pH=7) and 10% mobile phase B(acetonitrile) was loaded to wash out lines. Then, gradient was operatedfrom 10% B to 30% B in 80 minutes. Fractions were collected at recordedintervals when main peak began to elute. The purity of each fraction wasmonitored by analytical RP-HPLC. Fractions of purity <95% (not meetingthe Main Pool criteria) were pooled accordingly and forward processedusing the same buffer system and gradient elution parameters statedabove. All fractions with purity >95% were pooled and stored at 2-8° C.The purified peptide solution was diluted in a 1:1 ratio with purifiedwater and then loaded to the same RP-HPLC column. The counter-ionexchange was accomplished by washing the column with 2-3 column volumesof 0.5M ammonium acetate aqueous, followed by gradient elution from 90%C (0.2% AcOH aqueous solution) and 10% mobile phase D (ACN) to 50%mobile phase C and 50% mobile phase D in 50 minutes. Fractions werecollected at recorded intervals and monitored by analytical RP-HPLC. Thefractions (≧95%) were collected and lyophilized to obtain final drypeptide, 68.0 g (96.1% pure).

Example 5

Desalination and Isolation of SP-304 after purification by RP-HPLC

After plecanatide was purified by RP-HPLC as described in Example 4, itwas desalinated and isolated. Briefly, the purified plecanatide inammonium acetate/acetonitrile/water buffer was loaded onto a columnpacked with polymeric absorbent (macroporous adsorption resin) and theneluted by a mixture of alcohol/water. Finally, the peptide alcoholsolution was concentrated under reduced pressure, precipitated with anether, e.g., diethyl ether or MTBE, and dried under vacuum to give thefinal product.

Resin (Polymeric Adsorbents) Screening

Resin pre-treatment: Polymeric adsorbents, DA201-C (from Jiangsu Suqing,China; crosslinked polystyrene; surface area 1200-1400 m/g; average porediameter: 3-4 nm; pore volume: 1.1-1.2 ml/g; bulk density: 0.68-0.75g/ml; specific density: 1.03-1.1 g/ml; moisture: 50-60%; particle size:0.315-1.25 mm≧95%; effective diameter: 0.4-0.7 mm; uniformitycoefficient: ≦1.6%), DA201-H (from Jiangsu Suqing, China; crosslinkedpolystyrene; surface area ≧800 m²/g; average pore diameter: 6-8 nm; porevolume: 1.5-1.8 ml/g; bulk density: 0.65-0.70 g/ml; specific density:1.02-1.07 g/ml; moisture: 55-65%; particle size: 0.315-1.25 mm ≧95%;effective diameter: 0.4-0.7 mm; uniformity coefficient: ≦1.6% ), ADS-5(from Nankai Hecheng, China; crosslinked polystyrene; surface area520-600 m²/g; average pore diameter: 25-30 nm; bulk density: 0.7-0.8g/ml; moisture: 60-70%; particle size: 0.315˜1.25 mm≧95%; uniformitycoefficient: ≦1.6% ), and ADS-8 (from Nankai Hecheng, China; crosslinkedpolystyrene; surface area 450-500 m2/g; average pore diameter: 12-16 nm;bulk density: 0.65-0.75 g/ml; moisture: 60-70%; particle size:0.315˜1.25 mm≧95%; uniformity coefficient: <1.6%) were suspended in 4-6volume of ethanol overnight. Decant or suction off the supernatant fromthe settled resin. Add 6-8 volume of deionized water and resuspend theresin with gentle overhead stirring. Again, decant or suction off thesupernatant from the settled resin. Repeat the above water treatment anddecantation steps until fines appearance is minimal.

Column packing and regeneration: Resuspend the pre-treated resins abovewith 1-2 volume of deionized water to form the resin slurry respectivelyby using gentle agitation. Pour the resin slurry slowly down the insideof the column to prevent air entrapment. After the resin slurry has beenfully transferred to the column, rinse the inside of the column using asquirt bottle containing deionized water. Open the column outlet to froma settled resin bed (ID=4cm, H=10cm). Then the resin beds were washedsuccessively at a flow rate of 3CV per hour by 4 CV of deionized water,2CV of 5% HC1 aq, 4CV of deionized water, 2CV of 2% NaOH aq and finally4 CV of deionized water till the pH of the elute was around 7.

Preparation of loading samples: 2000 mg of lyophilized plecanatide wasdissolved in a mixture of 60 mL of ACN and 150 mL of 0.2% AcOH aq (thepH of the AcOH aq was adjusted to 4 with 10% ammonia aq.). Afterfiltration with 1.2 μm Nylon membrane, the filtrate was diluted to 250mL with 0.2% AcOH aq (pH4) and split into 4 parts (62.5 mL each) forloading.

Loading the sample to the columns: 62.5 mL of peptide solution above wasloaded onto the above 4 columns at a flow rate of 2 CV/h respectively.The loading elute was collected and tested by RP-HPLC to evaluate theabsorbent capacity of each resin. The absorbent capacity results of eachresin were demonstrated in Table X below.

TABLE X Peptide in loading Peptide Absorbent Resins sample absorbedratio DA201-C 500 mg 303.5 mg 60.7% DA201-H 500 mg 493.4 mg 98.7% ADS-5500 mg 450.5 mg 90.1% ADS-8 500 mg 466.1 mg 93.2%

HPLC Method: HPLC machine: Shimadzu LC-10AD vp; column: Kromasil, C18,4.6×250 mm ; mobile phase A: 0.1% TFA in water; mobile phase B: 0.1%TFAin ACN; detect at: 215 nm; column temperature: 40° C.; flow rate: 1.0mL/min; gradient: 25%B to 45%B in 30 min.

Absorbent Capacity Calculation: The absorbent capacity of each resin wasdemonstrated by the absorbent ratio of the peptide loaded onto eachcolumn, which was calculated by the quantity of the peptide absorbed ineach resin column divided by the peptide quantity in each loading sample(500 mg). The quantity of peptide absorbed in each column was calculatedby the formula below:

Quantity of peptide absorbed=Quantity of peptide in the loadingsample−Quantity of peptide in the loading elute=500 mg−62.5 mL×(1.6mg/mL×HPLC peak area of the eluate/HPLC peak area of the peptidestandard solution)

Washing the Column with Deionized Water: The loaded columns above werethen washed with 2 V of deionized water at 2 CV/h to remove the salts.The washing eluates were collected and analyzed by RP-HPLC to determinethe peptide quantity desorbed by water using the same method above. Thedesorbed peptide ratios of each resin were listed in Table XI below.

TABLE XI Peptide absorbed Peptide Desorption Resins in resin desorbedratio DA201-C 303.5 mg 184.5 mg  60.8% DA201-H 493.4 mg 40.9 mg 8.3%ADS-5 450.5 mg 41.9 mg 9.3% ADS-8 466.1 mg 40.1 mg 8.6%

Desorbing the Peptide with 90% Ethanol/Water: After 2 CV of waterwashes, the peptide absorbed in each column was then eluted by 1-2 CV of90% ethanol in water at 2 CV/h. The elution was collected and analyzedby RP-HPLC to determine the peptide quantity desorbed by 90%) ethanolusing the same method above. The desorbed peptide ratios of each resinwere listed in Table XII below.

TABLE XII Peptide absorbed Desorp- in resin after 2CV tion Resins waterwashes Peptide desorbed ratio DA201-C 119.0 mg 47.6 mg (by 2CV ethanol)  40% DA201-H 452.5 mg 452.5 mg (by 1.5CV ethanol) 100% ADS-5 408.6 mg408.6 mg (by 1.5CV ethanol) 100% ADS-8 426.0 mg 426.0 mg (by 1.5CVethanol) 100%

Isolation of the Peptide from the Ethanol Solution: The collectedpeptide/ethanol/water solution from each column was concentrated underreduced pressure, precipitated with MTBE, filtered and dried in vacuo togive the final product. The overall yield of the peptide processed byeach column was demonstrated in Table XIII below.

TABLE XIII Peptide in loading Final product Overall Resins sampleobtained yield DA201-C 500 mg  52 mg 10.4% DA201-H 500 mg 460 mg 92.0%ADS-5 500 mg 400 mg 80.0% ADS-8 500 mg 430 mg 86.0%

Resin Screening Conclusion: From the data above (Table X to Table XIII),the DA201-H resin presented the best absorbent capacity and the bestdesorption (by ethanol) performance for plecanatide among the resins inthe experiment.

Eluting Solvents Selection: Isopropanol and ethanol are two commonlyused solvents for eluting the peptide from the polymeric absorbents.Table XIV shows the amount of plecanatide that is able to be dissolvedin aqueous ethanol or isopropanol solution depending on the v/v %isopropanol (or ethanol): water.

TABLE XIV Solvent Solubility 90% IPA/Water  67.5 mg/mL 75% IPA/Water596.1 mg/mL 50% IPA/Water 635.0 mg/mL 90% EtOH/Water 302.0 mg/mL 75%EtOH/Water 700.0 mg/mL

The water in the peptide/alcohol solution can be removed by azeotropicdistillation. Table XV shows the property of the binary azeotropes ofethanol/water and isopropanol/water.

TABLE XV Azeotrope Component Component Boiling Boiling Boiling AzeotropeA B Point A Point B Point Wt. % A Water Ethanol 100° C. 78.3° C. 78.2°C.   4% Water Isopropanol 100° C. 82.3° C. 80.3° C. 12.6%

Degradation of plecanatide will occur during the long time storage ofthe peptide/alcohol/water solution and the concentration process. TableXVI demonstrates the stability data of plecanatide in 75% IPA/watersolution and 90% EtOH/water at 23° C.

TABLE XVI Duration Purity (in 90% EtOH aq.)* Purity (in 75% IPA aq.)* 0hours 98.5% 98.7% 2 hours 98.4% 98.7% 4 hours N/A 98.3% 6 hours 98.1%97.5% 8 hours N/A 96.6% 10 hours  95.5% N/A 24 hours  92.0% N/A 25hours  N/A 96.1% *HPLC Method: HPLC machine: Shimadzu LC-10AD vp:column: Kromasil, C18, 4.6 × 250 mm; mobile phase A: 0.1% TFA in water;mobile phase B: 0.1% TFA in ACN; detect at: 215 nm; column temperature:40° C.; flow rate: 1.0 mL/min; gradient: 25% B to 45% B in 30 min.

From the testing data obtained, plecanatide was fairly stable at 23° C.in alcohol/water solution within 6 hours.

Elution experiments were conducted using isopropanol/water mixtures withdifferent v/v %. The desorption ratio and water content of the peptideeluates were listed in Table XVII below.

TABLE XVII v/v % IPA/Water 75% 95% 100% IPA/water IPA/water IPA/waterWater content in peptide 65.9% 53.0% 49.8% Desorption ratio  100%  100% 90%

75% Isopropanol/water as eluting solution: 500 mg of plecanatide (98.1 %pure) was dissolved in a mixture of 16 mL ACN and 49 mL of 0.2% AcOHaqueous (the pH of the 0.2% AcOH solution was adjusted to 4.0 byaddition of 10% NH₄OH aq). After filtration by 1.2 μm nylon membrane,the peptide solution was loaded onto a column packed with DA201-H resin(ID=4 cm, H=10 cm, packed and p re-treated by the procedure mentionedpreviously) at 2 CV/h. After loading, the column was washed with 2 CVdeionized water at 2 CV/h. Then, the peptide was eluted by 1.5 CV of 75%IPA/water at 2 CV/h. The elution was monitored by RP-HPLC. The eluatewas collected (124 mL, 98.3% pure). Karl Fisher analysis indicated water(65.9% wt. %). Into a 1-neck, 500-mL round bottom flask was placed 124mL of peptide/IPA/water eluate collected above (97.6% pure, slightdegradation occurred during storage at 2-8° C. for 2 days). The flaskwas then placed under reduced pressure (60 Pa) on a rotary evaporatorand partially immersed in a 23° C. water bath. A whitish suspension wasformed by feed-stripping approximately 622 mL of isopropanol in 2 hoursto about ⅓ of the initial volume of the solution (97.7% pure). KarlFisher analysis indicated water (0.17% wt. %). To the concentrate wasadded 350 mL of pre-chilled diethyl ether, the solid was collected bycentrifugation at 3500 rpm for 3 minutes and dried under vacuum to yield333 mg of final product (yield 66.6%, 97.2% HPLC purity).

95% Isopropanol/water as eluting solution I: 500 mg of plecanatide(98.1% pure) was dissolved in a mixture of 16 mL ACN and 49 mL of 0.2%AcOH aqueous (the pH of the 0.2% AcOH solution was adjusted to 4.0 byaddition of 10% NH4OH aq). After filtration by 1.2 μm nylon membrane,the peptide solution was loaded onto a column packed with DA201-H resin(ID=4 cm, H=10 cm, pre-treated by the procedure mentioned previously) at2 CV/h. After loading, the column was washed with 2 CV deionized waterat 2 CV/h. Then, the peptide was eluted by 1.5CV of 95% IPA/water at 2CV/h. The elution was monitored by RP-HPLC. The eluate was collected(117 mL, 98.1 % pure). Karl Fisher analysis indicated water (52% wt. %).Into a 1-neck, 500-mL round bottom flask was placed 117 mL ofpeptide/IPA/water eluate collected above. The flask was then placedunder reduced pressure (50 Pa) on a rotary evaporator and partiallyimmersed in a 23° C. water bath. A whitish solid was formed byfeed-stripping approximately 470 mL of isopropanol in 130 min (97.9%pure). To the solid above was added 50 mL of pre-chilled diethyl etherto form a suspension and evaporated under reduced pressure (50 Pa) on arotary evaporator at 23° C. to dryness. Yield was 430 mg of finalproduct (86%). HPLC purity was 97.9%.

95% Isopropanol/water as eluting solution II: 500 mg of plecanatide(98.1 % pure) was dissolved in a mixture of 16 mL ACN and 49 mL of 0.2%)AcOH aqueous (the pH of the 0.2% AcOH solution was adjusted to 4.0 byaddition of 10% NH₄OH aq). After filtration by 1.2 μm nylon membrane,the peptide solution was loaded onto a column packed with DA201-H resin(ID=4 cm, H=10 cm, packed and pre-treated by the procedure mentionedpreviously) at 2 CV/h. After loading, the column was washed with 2 CVdeionized water at 2 CV/h. Then, the peptide was eluted by 1.5 CV of 95%IPA/water at 2 CV/h. The elution was monitored by RP-HPLC. The eluatewas collected (118 mL, 98.2% pure). Karl Fisher analysis indicated water(53% wt. %). Into a 1-neck, 500-mL round bottom flask was placed 118 mLof peptide/IPA/water eluate collected above. The flask was then placedunder reduced pressure (50 Pa) on a rotary evaporator and partiallyimmersed in a 23° C. water bath. A whitish suspension (˜40 mL) wasformed by feed-stripping approximately 330 mL of isopropanol in 100 min(97.7% pure). To the suspension above was added 400 mL of pre-chilleddiethyl ether to form a suspension. After standing at ambienttemperature for 1 hour, the solid was collected by centrifugation at3500 rpm for 3 minutes, and dried under vacuum to yield 370 mg of finalproduct. Yield was 74%. HPLC purity was 97.9%.

95% Isopropanol/water as eluting solution III: 10 g of plecanatide wasdesalted and precipitated in a manner similar to that described above.Interestingly, the precipitation yield was improved to 93%), which is asignificant increase in yield. HPLC purity after precipitation was98.47%.

Example 6 Characterization of Lyophilized SP-304 and Precipitated SP-304

The plecanatide purified by lyophilization as described in Example 4 andplecanatide purified by precipitation as described in Example 5 wereanalyzed to determine significant chemical impurity values such as thelevels of acetamide, TFA, ammonium ion, and acetonitrile. The resultsare listed in the table below.

Acetamide TFA Ammonium ion Acetonitrile Lyophilized 356 ppm 0.14% 1.58%40 ppm plecanatide Precipitated <28 ppm (LOQ 0.09% 0.23% Not Detectedplecanatide of method) (20 ppm LOQ)

As demonstrated by results above, the precipitation process providedsignificantly reduced levels of undesirable process impurities.

The plecanatide purified by lyophilization as described in Example 4 andplecanatide purified by precipitation as described in Example 5 weremeasured to obtain their bulk densities, tap densities, particle sizedistribution, and shape.

EQUIPMENT: (1) Tap Density Tester Model TD-1020; (2) Sonic SifterSeparator Model L3P; (3) Optical Microscope LINITRON 2850.

METHODS:

1) Bulk and Tap Density Measurements: Modified USP 1 Method

-   -   a. 100.0 mL graduate cylinder was used for lyophilized        plecanatide    -   b. 10.0 mL graduated cylinder was used for precipitated        plecanatide

2) Particle Size Distribution Analysis

-   -   a. Screens used: 200, 140, 100, 60, 40 and 30 meshes.    -   b. Sample size: 2 g of lyophilized plecanatide and 6.4 g of        precipitated plecanatide

3) Optical Microscopic Analysis: Particle Size and Shape

-   -   a. Dry powder was manually dispersed onto a microscopic plate    -   b. Magnification: 100 ×    -   c. Under normal light condition (no polarized filters)

RESULTS

(1 ) Physical Appearance: lyophilized plecanatide is a light, fluffy andwhite powder. Precipitated plecanatide is a slightly off-white powder.

(2) Bulk and Tap Density: Table XVIII provides bulk and tap density datafor Plecanatide samples of both lyophilized and precipitated:

TABLE XVIII Plecanatide Sample Bulk Density, g/mL Tap Density, g/mLLyophilized, Lot 101221 0.0332 0.0680 Precipitated, Lot 120210 0.4860.641

As seen from the data, the precipitated plecanatide is unexpectedlysignificantly denser than the lyophilized plecanatide. The precipitatedplecanatide has less tendency of dust generation during processing,which affords the advantages of increased safety and reduced processinglosses.

(3) Particle Size Distribution: Table XIX summarizes the particle sizedistribution analysis. FIG. 1 presents the data graphically.

TABLE XIX # Particle Mesh Size Weight Retained (g) Percent Retained Size(μm) Lyophilized Precipitated Lyophilized Precipitated 30 600 0.07 0.453.6% 7.1% 40 425 0.26 0.17 13.3% 2.7% 60 250 0.17 0.67 8.7% 10.5% 100150 0.51 2.08 26.0% 32.7% 140 106 0.65 1.22 33.2% 19.2% 200 75 0.24 0.6812.2% 10.7% Pan <75 0.06 1.09 3.1% 17.1% Total 1.96 6.36 100.0% 100.0%

As demonstrated by Table XIX and FIG. 1 , the particle sizedistributions are different for both types of plecanatide. Duringanalysis, it was observed that the precipitated plecanatide containedsome larger particles, which could be broken up easily. It was alsonoticed that the lyophilized plecanatide appeared to be flaky andsticking onto top and bottom of sieves, whereas no sticking was observedfor the precipitated plecanatide. It indicates a better processingproperty of the precipitated plecanatide.

(4) Particle Size and Shape; FIGS. 2 and 3 provide optical microscopicanalysis of samples of lyophilized and precipitated plecanatide,respectively. As seen in FIG. 2, the lyophilized plecanatide is inamorphous form and has irregular shapes of particles. They form physicalaggregates with particles lying on top of each other. In FIG. 3, theprecipitated plecanatide shows distinguishable individual particles.From the particle appearances and shapes, the precipitated plecanatidewill have better flow property and therefore can facilitate solidprocessing during manufacturing.

The lyophilized and precipitated forms of plecanatide have showndistinguishable physical appearances and properties by density, particlesize distribution and shape analyses. The precipitated form is moresuitable for solid dosage form processing during manufacturing in termsof reducing dust generation, less sticking onto processing equipment,and potentially less processing losses.

The precipitated form is more suitable for processing solid dosage formduring manufacture (e.g., a low-dose solid dosage form). The higherdensity of the precipitated material will reduce aerosol or “dust”losses during weighing, transferring, and blending. The differentparticle shape has been shown to reduce loss caused by sticking toscreens or sieves. The higher density should improve content uniformitysince the size and density of the drug particles more closely matchthose of the excipients.

Example 7 Low-Dose Formulation of Precipitated SP-304

The plecanatide purified by precipitation as described in Example 5 isprocessed further to make low-dose formulations as described below.

composition of dry-blending batch Concentration % Item No. Ingredientw/w 1 SP-304 0.3246 2 Microcrystalline 99.43 cellulose (Avicel PH 102) 3Magnesium stearate 0.2500 4 HPMC capsule shells n/a Total 100

Blending:

Avicel PH 102 is screened through a 60 mesh screen. V-blenders (1 Qt, 4Qt, and 16 Qt) are then dusted by the screened Avicel PH 102. SP-304 isscreened through a 200 mesh screen and loaded into the 1-Qt V-blender.Then, about 80 g Avicel PH 102 is added into the 1-Qt blender and themixture is blended for 10 minutes at 25 rpm. The mixture is thentransferred to the 4-Qt V-blender which is pre-dusted by the screenedAvicel PH 102. The 1-Qt blender is rinsed with Avicel and the rinsematerial is transferred to the 4-Qt blender. The rinsing is repeateduntil all SP-304 is transferred to the 4-Qt blender. About 200 g Avicelis added to the 4-Qt V-blender and the mixture is blended for 10minutes. The resulting blend is then screened through a 60 mesh screenand then transferred into the pre-dusted 16-Qt blender (dusted with 1500g Avicel). The 4-Qt blender is rinsed with Avicel and the rinse materialis transferred to the 16-Qt blender. The remaining Avicel is added tothe 16-Qt blender and the mixture is blended for 10 minutes. Theresulting blend is passed through Comil, rinsed with excess of Avicel,and then returned to the 16-Qt blender and is further blended for 5minutes. Proper amount of magnesium stearate is weighed, screenedthrough a 60 mesh screen, and added into the 16-Qt blender. Theresulting mixture is blended for 2 minutes. The resulting mixture isthen either packaged in capsules or compressed to form tablets.

Encapsulation

A MG2 Planeta capsule filler is set up. Average weight of the emptycapsule shells is determined and target capsule fill weight wascalculated (±5%). The blend from the above process is added into thehopper of the capsule filler and encapsulation is started. Run weightparameters are manually adjusted. Resulting capsules are then sortedaccording to the target fill weight.

Compression

A Fette tablet press is set up. Then the blend mixture is loaded intothe powder hopper and tooling is installed. The weight of each tablet isset to be 100 mg±5% and hardness to be 4-6 Kp. The weight, hardness, andthickness of tablets are measured and recorded every 5 to 10 minutes.Friability measurement is also performed to ensure satisfactory product.

1-51. (canceled)
 52. A purified peptide comprising the GCC agonistsequence of SEQ ID NO: 1, wherein the peptide has a bulk density of noless than 0.1 g/mL, no less than 0.2 g/mL, no less than 0.3 g/mL, noless than 0.4 g/mL, or no less than 0.5 g/mL.
 53. The purified peptideof claim 52, wherein the peptide has a tap density of no less than 0.1g/mL, no less than 0.2 g/mL, no less than 0.3 g/mL, no less than 0.4g/mL, no less than 0.5 g/mL, or no less than 0.6 g/mL.
 54. The purifiedpeptide of claim 52, wherein the peptide has a chromatographic purity ofno less than 95%, no less than 97%, or no less than 98%.
 55. Thepurified peptide of claim 54, wherein the average total impurities isless than 3%.
 56. The purified peptide of claim 55, wherein peptide issubstantially free of impurities selected from acetonitrile, acetamide,alcohols, ammonium, and TFA.
 57. The purified peptide of claim 52,wherein peptide is substantially free of water.
 58. The purified peptideof claim 57, wherein the average water content of the peptide is lessthan 10% (w/w).
 59. The purified peptide of claim 52, wherein thepeptide is substantially free of topoisomers.
 60. The purified peptideof claim 52, wherein the approximate yield of the peptide is greaterthan 15% (w/w).